Links to Exploration step
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice</title><author><name sortKey="Tomimatsu, Nozomi" sort="Tomimatsu, Nozomi" uniqKey="Tomimatsu N" first="Nozomi" last="Tomimatsu">Nozomi Tomimatsu</name><affiliation><nlm:aff id="A1"> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</nlm:aff></affiliation></author><author><name sortKey="Mukherjee, Bipasha" sort="Mukherjee, Bipasha" uniqKey="Mukherjee B" first="Bipasha" last="Mukherjee">Bipasha Mukherjee</name><affiliation><nlm:aff id="A1"> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</nlm:aff></affiliation></author><author><name sortKey="Hardebeck, Molly Catherine" sort="Hardebeck, Molly Catherine" uniqKey="Hardebeck M" first="Molly Catherine" last="Hardebeck">Molly Catherine Hardebeck</name><affiliation><nlm:aff id="A1"> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</nlm:aff></affiliation></author><author><name sortKey="Ilcheva, Mariya" sort="Ilcheva, Mariya" uniqKey="Ilcheva M" first="Mariya" last="Ilcheva">Mariya Ilcheva</name><affiliation><nlm:aff id="A1"> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</nlm:aff></affiliation></author><author><name sortKey="Camacho, Cristel Vanessa" sort="Camacho, Cristel Vanessa" uniqKey="Camacho C" first="Cristel Vanessa" last="Camacho">Cristel Vanessa Camacho</name><affiliation><nlm:aff id="A1"> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</nlm:aff></affiliation></author><author><name sortKey="Harris, Janelle Louise" sort="Harris, Janelle Louise" uniqKey="Harris J" first="Janelle Louise" last="Harris">Janelle Louise Harris</name><affiliation><nlm:aff id="A2">Signal Transduction Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4029, Australia</nlm:aff></affiliation></author><author><name sortKey="Porteus, Matthew" sort="Porteus, Matthew" uniqKey="Porteus M" first="Matthew" last="Porteus">Matthew Porteus</name><affiliation><nlm:aff id="A3">Deparment of Pediatrics, Stanford University, Stanford, CA 94395, USA</nlm:aff></affiliation></author><author><name sortKey="Llorente, Bertrand" sort="Llorente, Bertrand" uniqKey="Llorente B" first="Bertrand" last="Llorente">Bertrand Llorente</name><affiliation><nlm:aff id="A4">CRCM, Inserm, U1068; Institut Paoli-Calmettes; Aix-Marseille Université, UM 105; CNRS, UMR7258, F-13009, Marseille, France</nlm:aff></affiliation></author><author><name sortKey="Khanna, Kum Kum" sort="Khanna, Kum Kum" uniqKey="Khanna K" first="Kum Kum" last="Khanna">Kum Kum Khanna</name><affiliation><nlm:aff id="A2">Signal Transduction Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4029, Australia</nlm:aff></affiliation></author><author><name sortKey="Burma, Sandeep" sort="Burma, Sandeep" uniqKey="Burma S" first="Sandeep" last="Burma">Sandeep Burma</name><affiliation><nlm:aff id="A1"> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24705021</idno><idno type="pmc">4041212</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041212</idno><idno type="RBID">PMC:4041212</idno><idno type="doi">10.1038/ncomms4561</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">002D86</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">002D86</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice</title><author><name sortKey="Tomimatsu, Nozomi" sort="Tomimatsu, Nozomi" uniqKey="Tomimatsu N" first="Nozomi" last="Tomimatsu">Nozomi Tomimatsu</name><affiliation><nlm:aff id="A1"> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</nlm:aff></affiliation></author><author><name sortKey="Mukherjee, Bipasha" sort="Mukherjee, Bipasha" uniqKey="Mukherjee B" first="Bipasha" last="Mukherjee">Bipasha Mukherjee</name><affiliation><nlm:aff id="A1"> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</nlm:aff></affiliation></author><author><name sortKey="Hardebeck, Molly Catherine" sort="Hardebeck, Molly Catherine" uniqKey="Hardebeck M" first="Molly Catherine" last="Hardebeck">Molly Catherine Hardebeck</name><affiliation><nlm:aff id="A1"> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</nlm:aff></affiliation></author><author><name sortKey="Ilcheva, Mariya" sort="Ilcheva, Mariya" uniqKey="Ilcheva M" first="Mariya" last="Ilcheva">Mariya Ilcheva</name><affiliation><nlm:aff id="A1"> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</nlm:aff></affiliation></author><author><name sortKey="Camacho, Cristel Vanessa" sort="Camacho, Cristel Vanessa" uniqKey="Camacho C" first="Cristel Vanessa" last="Camacho">Cristel Vanessa Camacho</name><affiliation><nlm:aff id="A1"> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</nlm:aff></affiliation></author><author><name sortKey="Harris, Janelle Louise" sort="Harris, Janelle Louise" uniqKey="Harris J" first="Janelle Louise" last="Harris">Janelle Louise Harris</name><affiliation><nlm:aff id="A2">Signal Transduction Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4029, Australia</nlm:aff></affiliation></author><author><name sortKey="Porteus, Matthew" sort="Porteus, Matthew" uniqKey="Porteus M" first="Matthew" last="Porteus">Matthew Porteus</name><affiliation><nlm:aff id="A3">Deparment of Pediatrics, Stanford University, Stanford, CA 94395, USA</nlm:aff></affiliation></author><author><name sortKey="Llorente, Bertrand" sort="Llorente, Bertrand" uniqKey="Llorente B" first="Bertrand" last="Llorente">Bertrand Llorente</name><affiliation><nlm:aff id="A4">CRCM, Inserm, U1068; Institut Paoli-Calmettes; Aix-Marseille Université, UM 105; CNRS, UMR7258, F-13009, Marseille, France</nlm:aff></affiliation></author><author><name sortKey="Khanna, Kum Kum" sort="Khanna, Kum Kum" uniqKey="Khanna K" first="Kum Kum" last="Khanna">Kum Kum Khanna</name><affiliation><nlm:aff id="A2">Signal Transduction Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4029, Australia</nlm:aff></affiliation></author><author><name sortKey="Burma, Sandeep" sort="Burma, Sandeep" uniqKey="Burma S" first="Sandeep" last="Burma">Sandeep Burma</name><affiliation><nlm:aff id="A1"> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</nlm:aff></affiliation></author></analytic><series><title level="j">Nature communications</title><idno type="eISSN">2041-1723</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="P2">Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. While CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival, and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favor HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In sum, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Wyman, C" uniqKey="Wyman C">C Wyman</name></author><author><name sortKey="Kanaar, R" uniqKey="Kanaar R">R Kanaar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Malumbres, M" uniqKey="Malumbres M">M Malumbres</name></author><author><name sortKey="Barbacid, M" uniqKey="Barbacid M">M Barbacid</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Symington, Ls" uniqKey="Symington L">LS Symington</name></author><author><name sortKey="Gautier, J" uniqKey="Gautier J">J Gautier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ferretti, Lp" uniqKey="Ferretti L">LP Ferretti</name></author><author><name sortKey="Lafranchi, L" uniqKey="Lafranchi L">L Lafranchi</name></author><author><name sortKey="Sartori, Aa" uniqKey="Sartori A">AA Sartori</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Huertas, P" uniqKey="Huertas P">P Huertas</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gravel, S" uniqKey="Gravel S">S Gravel</name></author><author><name sortKey="Chapman, Jr" uniqKey="Chapman J">JR Chapman</name></author><author><name sortKey="Magill, C" uniqKey="Magill C">C Magill</name></author><author><name sortKey="Jackson, Sp" uniqKey="Jackson S">SP Jackson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mimitou, Ep" uniqKey="Mimitou E">EP Mimitou</name></author><author><name sortKey="Symington, Ls" uniqKey="Symington L">LS Symington</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhu, Z" uniqKey="Zhu Z">Z Zhu</name></author><author><name sortKey="Chung, Wh" uniqKey="Chung W">WH Chung</name></author><author><name sortKey="Shim, Ey" uniqKey="Shim E">EY Shim</name></author><author><name sortKey="Lee, Se" uniqKey="Lee S">SE Lee</name></author><author><name sortKey="Ira, G" uniqKey="Ira G">G Ira</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nimonkar, Av" uniqKey="Nimonkar A">AV Nimonkar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Huertas, P" uniqKey="Huertas P">P Huertas</name></author><author><name sortKey="Jackson, Sp" uniqKey="Jackson S">SP Jackson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Huertas, P" uniqKey="Huertas P">P Huertas</name></author><author><name sortKey="Cortes Ledesma, F" uniqKey="Cortes Ledesma F">F Cortes-Ledesma</name></author><author><name sortKey="Sartori, Aa" uniqKey="Sartori A">AA Sartori</name></author><author><name sortKey="Aguilera, A" uniqKey="Aguilera A">A Aguilera</name></author><author><name sortKey="Jackson, Sp" uniqKey="Jackson S">SP Jackson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yun, Mh" uniqKey="Yun M">MH Yun</name></author><author><name sortKey="Hiom, K" uniqKey="Hiom K">K Hiom</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, L" uniqKey="Chen L">L Chen</name></author><author><name sortKey="Nievera, Cj" uniqKey="Nievera C">CJ Nievera</name></author><author><name sortKey="Lee, Ay" uniqKey="Lee A">AY Lee</name></author><author><name sortKey="Wu, X" uniqKey="Wu X">X Wu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, H" uniqKey="Wang H">H Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Buis, J" uniqKey="Buis J">J Buis</name></author><author><name sortKey="Stoneham, T" uniqKey="Stoneham T">T Stoneham</name></author><author><name sortKey="Spehalski, E" uniqKey="Spehalski E">E Spehalski</name></author><author><name sortKey="Ferguson, Do" uniqKey="Ferguson D">DO Ferguson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Falck, J" uniqKey="Falck J">J Falck</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bolderson, E" uniqKey="Bolderson E">E Bolderson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Costelloe, T" uniqKey="Costelloe T">T Costelloe</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tomimatsu, N" uniqKey="Tomimatsu N">N Tomimatsu</name></author><author><name sortKey="Mukherjee, B" uniqKey="Mukherjee B">B Mukherjee</name></author><author><name sortKey="Burma, S" uniqKey="Burma S">S Burma</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tomimatsu, N" uniqKey="Tomimatsu N">N Tomimatsu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Byth, Kf" uniqKey="Byth K">KF Byth</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Meijer, L" uniqKey="Meijer L">L Meijer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Stevenson Lindert, Lm" uniqKey="Stevenson Lindert L">LM Stevenson-Lindert</name></author><author><name sortKey="Fowler, P" uniqKey="Fowler P">P Fowler</name></author><author><name sortKey="Lew, J" uniqKey="Lew J">J Lew</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Orans, J" uniqKey="Orans J">J Orans</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, Rq" uniqKey="Chen R">RQ Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="El Shemerly, M" uniqKey="El Shemerly M">M El-Shemerly</name></author><author><name sortKey="Hess, D" uniqKey="Hess D">D Hess</name></author><author><name sortKey="Pyakurel, Ak" uniqKey="Pyakurel A">AK Pyakurel</name></author><author><name sortKey="Moselhy, S" uniqKey="Moselhy S">S Moselhy</name></author><author><name sortKey="Ferrari, S" uniqKey="Ferrari S">S Ferrari</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shiromizu, T" uniqKey="Shiromizu T">T Shiromizu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bekker Jensen, S" uniqKey="Bekker Jensen S">S Bekker-Jensen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Takeda, Dy" uniqKey="Takeda D">DY Takeda</name></author><author><name sortKey="Wohlschlegel, Ja" uniqKey="Wohlschlegel J">JA Wohlschlegel</name></author><author><name sortKey="Dutta, A" uniqKey="Dutta A">A Dutta</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Eid, W" uniqKey="Eid W">W Eid</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Huen, Ms" uniqKey="Huen M">MS Huen</name></author><author><name sortKey="Sy, Sm" uniqKey="Sy S">SM Sy</name></author><author><name sortKey="Chen, J" uniqKey="Chen J">J Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Greenberg, Ra" uniqKey="Greenberg R">RA Greenberg</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yu, X" uniqKey="Yu X">X Yu</name></author><author><name sortKey="Chen, J" uniqKey="Chen J">J Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bunting, Sf" uniqKey="Bunting S">SF Bunting</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mukherjee, B" uniqKey="Mukherjee B">B Mukherjee</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tran, Pt" uniqKey="Tran P">PT Tran</name></author><author><name sortKey="Erdeniz, N" uniqKey="Erdeniz N">N Erdeniz</name></author><author><name sortKey="Symington, Ls" uniqKey="Symington L">LS Symington</name></author><author><name sortKey="Liskay, Rm" uniqKey="Liskay R">RM Liskay</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Andersen, Sd" uniqKey="Andersen S">SD Andersen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Liberti, Se" uniqKey="Liberti S">SE Liberti</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Peterson, Se" uniqKey="Peterson S">SE Peterson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yu, X" uniqKey="Yu X">X Yu</name></author><author><name sortKey="Fu, S" uniqKey="Fu S">S Fu</name></author><author><name sortKey="Lai, M" uniqKey="Lai M">M Lai</name></author><author><name sortKey="Baer, R" uniqKey="Baer R">R Baer</name></author><author><name sortKey="Chen, J" uniqKey="Chen J">J Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Daley, Jm" uniqKey="Daley J">JM Daley</name></author><author><name sortKey="Sung, P" uniqKey="Sung P">P Sung</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nicolette, Ml" uniqKey="Nicolette M">ML Nicolette</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, X" uniqKey="Chen X">X Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cuadrado, M" uniqKey="Cuadrado M">M Cuadrado</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mcellin, B" uniqKey="Mcellin B">B McEllin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Moyal, L" uniqKey="Moyal L">L Moyal</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<pmc-dir>properties manuscript</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-journal-id">101528555</journal-id><journal-id journal-id-type="pubmed-jr-id">37539</journal-id><journal-id journal-id-type="nlm-ta">Nat Commun</journal-id><journal-id journal-id-type="iso-abbrev">Nat Commun</journal-id><journal-title-group><journal-title>Nature communications</journal-title></journal-title-group><issn pub-type="epub">2041-1723</issn></journal-meta><article-meta><article-id pub-id-type="pmid">24705021</article-id><article-id pub-id-type="pmc">4041212</article-id><article-id pub-id-type="doi">10.1038/ncomms4561</article-id><article-id pub-id-type="manuscript">NIHMS573163</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Tomimatsu</surname><given-names>Nozomi</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Mukherjee</surname><given-names>Bipasha</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Hardebeck</surname><given-names>Molly Catherine</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ilcheva</surname><given-names>Mariya</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Camacho</surname><given-names>Cristel Vanessa</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="author-notes" rid="FN1">2</xref></contrib><contrib contrib-type="author"><name><surname>Harris</surname><given-names>Janelle Louise</given-names></name><xref ref-type="aff" rid="A2">3</xref></contrib><contrib contrib-type="author"><name><surname>Porteus</surname><given-names>Matthew</given-names></name><xref ref-type="aff" rid="A3">4</xref></contrib><contrib contrib-type="author"><name><surname>Llorente</surname><given-names>Bertrand</given-names></name><xref ref-type="aff" rid="A4">5</xref></contrib><contrib contrib-type="author"><name><surname>Khanna</surname><given-names>Kum Kum</given-names></name><xref ref-type="aff" rid="A2">3</xref></contrib><contrib contrib-type="author"><name><surname>Burma</surname><given-names>Sandeep</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="corresp" rid="CR1">*</xref></contrib></contrib-group><aff id="A1"><label>1</label> Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390, USA</aff><aff id="A2"><label>3</label>Signal Transduction Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4029, Australia</aff><aff id="A3"><label>4</label>Deparment of Pediatrics, Stanford University, Stanford, CA 94395, USA</aff><aff id="A4"><label>5</label>CRCM, Inserm, U1068; Institut Paoli-Calmettes; Aix-Marseille Université, UM 105; CNRS, UMR7258, F-13009, Marseille, France</aff><author-notes><fn fn-type="present-address" id="FN1"><label>2</label><p id="P1">Current Address: Department of Genetics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA</p></fn><corresp id="CR1"><label>*</label>Address correspondence to: Sandeep Burma, Tel: 214-648-7440; Fax: 214-648-5995; <email>sandeep.burma@utsouthwestern.edu</email></corresp></author-notes><pub-date pub-type="nihms-submitted"><day>21</day><month>5</month><year>2014</year></pub-date><pub-date pub-type="epub"><day>07</day><month>4</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><pub-date pub-type="pmc-release"><day>07</day><month>10</month><year>2014</year></pub-date><volume>5</volume><fpage>3561</fpage><lpage>3561</lpage><pmc-comment>elocation-id from pubmed: 10.1038/ncomms4561</pmc-comment>
<permissions><license xlink:href="http://www.nature.com/authors/editorial_policies/license.html#terms"><license-p>Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:<ext-link ext-link-type="uri" xlink:href="http://www.nature.com/authors/editorial_policies/license.html#terms">http://www.nature.com/authors/editorial_policies/license.html#terms</ext-link></license-p></license></permissions><abstract><p id="P2">Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. While CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival, and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favor HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In sum, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice.</p></abstract><kwd-group><kwd>DNA repair</kwd><kwd>DNA end resection</kwd><kwd>repair pathway choice</kwd><kwd>homologous recombination</kwd><kwd>EXO1</kwd><kwd>CDK</kwd><kwd>DNA damage response</kwd></kwd-group></article-meta></front><body><sec sec-type="intro" id="S1"><title>INTRODUCTION</title><p id="P3">The integrity of our genome is constantly threatened by DNA double-strand breaks (DSBs). Two major pathways have evolved to repair these breaks<sup><xref rid="R1" ref-type="bibr">1</xref></sup>. Non-homologous end joining (NHEJ) is an error-prone pathway during which DNA ends are ligated independently of a template, whereas homologous recombination (HR) is an error-free pathway that uses the intact sister chromatid as a template for repair. Cell cycle-regulation of these two pathways is critical for the maintenance of genomic integrity. Suppression of HR in G1 prevents loss of heterozygosity and chromosomal rearrangements arising from recombination with the homologous chromosome or non-allelic homologous loci, respectively. On the other hand, promotion of HR in S and G2 phases ensures accurate repair, especially that of one-ended replication fork-associated breaks. However, the underlying mechanisms of cell cycle regulation of NHEJ and HR pathways are not completely worked out.</p><p id="P4">Coupling DNA repair machineries to the engine that drives the cell cycle, namely cyclin-dependent kinases (CDKs) in complex with their cyclin partners<sup><xref rid="R2" ref-type="bibr">2</xref></sup>, is an efficient way to achieve cell cycle regulation of DSB repair. It is becoming increasingly clear that the CDKs that promote cell cycle progression through S and G2 phases concomitantly promote HR in these phases by activating the first step in recombination, viz. DNA end resection<sup><xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R4" ref-type="bibr">4</xref></sup>. DNA end resection in the 5’ to 3’ direction results in the formation of a 3’-OH single-stranded tail that is first coated with RPA which is then exchanged for Rad51 to form a recombinogenic nucleofilament. Resection is a key step at which “repair pathway choice”, the choice between NHEJ and HR, can be enforced by the cell as the generation of ssDNA would favor HR and concomitantly disfavor NHEJ by stymieing the binding of NHEJ proteins. A “two-step” model of resection has been formulated, based mainly upon studies in yeast<sup><xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R5" ref-type="bibr">5</xref></sup>. The first step, “initiation of resection”, involves the removal of ~50-100 bases of DNA from the 5’ end by the MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast and Mre11-Rad50-Nbs1 in humans) in conjunction with Sae2/CtIP<sup><xref rid="R6" ref-type="bibr">6</xref>-<xref rid="R9" ref-type="bibr">9</xref></sup>. The second step, “long-range resection”, is carried out by two alternate pathways involving the 5’ to 3’ exonuclease Exo1 on one hand, and the helicase-topoisomerase complex Sgs1-Top3-Rmi1/BLM-TOPIIIα-RMI1-2 in concert with the nuclease Dna2, on the other hand. The current understanding is that CDKs control DNA end resection and repair pathway choice by promoting the initiation of resection via phosphorylation of Sae2/CtIP and Nbs1<sup><xref rid="R10" ref-type="bibr">10</xref>-<xref rid="R16" ref-type="bibr">16</xref></sup>. Here, we describe a novel mechanism by which CDKs control repair pathway choice in human cells which involves regulation of long-range resection via phosphorylation of EXO1.</p></sec><sec sec-type="results" id="S2"><title>RESULTS</title><sec id="S3"><title>EXO1 is phosphorylated by CDKs 1 and 2 in S and G2 phases</title><p id="P5">We have previously shown that EXO1 plays a major role in resection and HR in human cells<sup><xref rid="R17" ref-type="bibr">17</xref>-<xref rid="R20" ref-type="bibr">20</xref></sup>, making it a plausible candidate for regulation by CDKs in a cell cycle-dependent manner. To examine this possibility, HEK-293 cells ectopically expressing V5-tagged EXO1 were synchronized in different phases of the cell cycle <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 1a</xref>)</bold>. EXO1 was immunoprecipitated (IP) with anti-V5 antibody and Western blotted with anti-phospho-(Ser) or anti-phospho-(Thr) CDK substrate antibodies. We observed that EXO1 was increasingly phosphorylated as cells traversed the cell cycle from G1, through S, to G2 <bold>(<xref ref-type="fig" rid="F1">Fig. 1a</xref>)</bold>. No increase in EXO1 phosphorylation was seen upon treating cells with ionizing radiation (IR) or UV indicating that the increase in EXO1 phosphorylation seen with cell cycle progression was not due to any genotoxic stress caused by cell synchronization methods <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 1b</xref>)</bold>. Phosphorylation of EXO1 in cells synchronized in G2 was attenuated by the CDK inhibitors AZD5438 and Roscovitine<sup><xref rid="R21" ref-type="bibr">21</xref>,<xref rid="R22" ref-type="bibr">22</xref></sup>, both of which inhibit CDKs 1 and 2, the principal CDKs driving progression through S and G2 phases<sup><xref rid="R2" ref-type="bibr">2</xref></sup><bold>(<xref ref-type="fig" rid="F1">Fig. 1a,b</xref>; <xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 1c</xref>)</bold>. Phosphorylation of EXO1 in cells synchronized in G2 was also attenuated upon siRNA-mediated depletion of CDKs 1 and 2 <bold>(<xref ref-type="fig" rid="F1">Fig. 1c</xref>; <xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 1d</xref>)</bold>. Depletion of CDK 1 and/or 2 did not significantly alter cell cycle profiles (<bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 1e</xref>)</bold>; hence the observed reduction in EXO1 phosphorylation was not due a reduction in the total population in S and G2. These observations indicated that EXO1 is hyperphosphorylated by CDKs 1 and 2 in S and G2 phases of the cell cycle and hinted that EXO1 may be regulated by CDKs.</p><p id="P6">Human EXO1 bears ten putative S/TP CDK phosphorylation sites, two of which (T732 and S815) conform to the optimal CDK-recognition motif, (S/T)PX(K/R)<sup><xref rid="R23" ref-type="bibr">23</xref></sup><bold>(<xref ref-type="fig" rid="F1">Fig. 1d</xref>)</bold>. To identify S/TP sites that might be important for resection, U2OS cells with siRNA-mediated depletion of endogenous EXO1 were complemented with ten different siRNA-resistant EXO1 constructs that were individually mutated to non-phosphorylatable Ala at each of these putative phosphorylation sites. siRNA-resistant wild type EXO1 (rWT-EXO1) served as a positive control, while siRNA-sensitive wild type EXO1 (sWT-EXO1) and nuclease dead EXO1 bearing a D173A mutation<sup><xref rid="R24" ref-type="bibr">24</xref></sup> (ND-EXO1) served as negative controls. Cells were screened for resection defects by quantifying RPA foci after irradiation, as described previously<sup><xref rid="R19" ref-type="bibr">19</xref></sup> (see legends to <bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 1f-h</xref></bold> for description of the screen and controls). We found that individual mutations of the four C-terminal S/TP sites (S639, T732, S815, and T824) resulted in resection defects as evidenced by reduced numbers of RPA foci <bold>(<xref ref-type="fig" rid="F1">Fig. 1e</xref>)</bold>. Mutation of all four sites to Ala (4AEXO1) eliminated EXO1 detection by anti-CDK substrate antibodies <bold>(<xref ref-type="fig" rid="F1">Fig. 1f</xref>)</bold>. However, negligible reduction was observed with individual mutations of these four sites probably because the anti-CDK substrate antibodies are not site-specific and would recognize multiple phosphorylated S/TP sites on the same protein <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 1i</xref>)</bold>. Therefore, in order to directly demonstrate phosphorylation of specific S/TP sites in vivo, we attempted to generate phospho-specific antibodies against these sites, and were successful in raising antibodies to phospho-T732 (<bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 1j,k</xref>)</bold>. In IP-Westerns, the anti-p-T732 antibody could recognize WT-EXO1 but not the non-phosphorylatable T732A mutant, confirming the specificity of this antibody <bold>(<xref ref-type="fig" rid="F1">Fig. 1g</xref>)</bold>. Phosphorylation of EXO1 at T732 was cell cycle- and CDK-dependent, with a higher level of phosphorylation observed in G2 that was attenuated upon pretreatment of cells with the CDK inhibitor AZD5438 <bold>(<xref ref-type="fig" rid="F1">Fig. 1g</xref>)</bold>. Akin to V5-tagged EXO1, endogenous EXO1 in these cells was also phosphorylated in a cell cycle- and CDK-dependent manner (<bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 1l</xref>)</bold>. Our results directly demonstrate that T732 of EXO1 is phosphorylated in vivo in a cell cycle- and CDK-dependent manner. Moreover, Western blotting results with 4A-EXO1 <bold>(<xref ref-type="fig" rid="F1">Fig. 1f</xref>)</bold> tentatively indicate that the other three identified sites are also phosphorylated in S/G2 phases, a supposition borne out by independent mass spectrometric analyses identifying S639 and S815 of EXO1 as bona fide phosphorylation targets in vivo<sup><xref rid="R25" ref-type="bibr">25</xref>-<xref rid="R27" ref-type="bibr">27</xref></sup>.</p></sec><sec id="S4"><title>Phosphorylation of EXO1 by CDKs promotes DNA end resection</title><p id="P7">To definitively establish the role of these four sites in regulation of DSB resection and HR, we utilized three complementary assays involving quantification of RPA, BrdU/ssDNA, and Rad51 foci, and compared foci formation in cells expressing WT-EXO1, non-phosphorylatable 4AEXO1, or phospho-mimic 4D-EXO1 (all four sites replaced with Asp). Cells expressing 4AEXO1 were severely impaired in RPA foci formation after irradiation, similar to that seen in cells expressing ND-EXO1 <bold>(<xref ref-type="fig" rid="F2">Fig. 2a</xref>; <xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 2a</xref>)</bold>, and this defect was more severe than that seen with EXO1 individually mutated at these four sites <bold>(<xref ref-type="fig" rid="F1">Fig. 1e</xref>)</bold>. Interestingly, cells expressing 4D-EXO1 were proficient in RPA foci formation and actually exhibited higher numbers of foci than cells expressing wild type EXO1 (rWT-EXO1). The impairment in RPA foci formation upon expression of 4A-EXO1 was clearly due to impaired DNA resection, directly quantified as IR-induced BrdU/ssDNA foci, as described previously<sup><xref rid="R20" ref-type="bibr">20</xref></sup><bold>(<xref ref-type="fig" rid="F2">Fig. 2b</xref>)</bold>. The resection defect in cells expressing 4A-EXO1 resulted in a decrease in IR-induced Rad51 foci<sup><xref rid="R17" ref-type="bibr">17</xref></sup> in S/G2 cells (Cyclin A-positive<sup><xref rid="R28" ref-type="bibr">28</xref></sup>) suggesting attenuated HR <bold>(<xref ref-type="fig" rid="F2">Fig. 2c</xref>)</bold>. The observed impairments in resection and HR were not due to perturbations of the cell cycle as assessed by flow cytometry <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 2b</xref>)</bold> or attenuation of the nuclease activity of 4A-EXO1 as assessed by in vitro nuclease assays <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 2c</xref>)</bold>.</p><p id="P8">Pharmacological inhibition of CDKs using AZD5438 or Roscovitine severely impaired resection and HR <bold>(<xref ref-type="fig" rid="F2">Fig. 2d-f</xref>; <xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 2d-f</xref>)</bold>. Remarkably, 4D-EXO1 could partially override the effects of CDK inhibition. In accord with these results, expression of 4D-EXO1 could partially rescue decreased IR-induced RPA foci formation in CtIP- or DNA2-depleted cells <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 2g,h</xref>)</bold> demonstrating that phosphorylation of EXO1 is sufficient to trigger substantial resection in human cells, and therefore, would be a step over which the cell must exercise stringent control as it traverses the cell cycle.</p></sec><sec id="S5"><title>Interaction of EXO1 and CDKs is obligatory for DNA end resection</title><p id="P9">CDK-cyclin complexes, especially CDK2-Cyclin A, bind to their substrates in a bipartite manner, with CDK binding to its phosphorylation site(s) and cyclin binding to distal RXL motif(s) called Cy<sup><xref rid="R23" ref-type="bibr">23</xref>,<xref rid="R29" ref-type="bibr">29</xref></sup>. We found that V5-EXO1 interacted with CDK1, CDK2, Cyclin A, and Cyclin B in co-IP experiments <bold>(<xref ref-type="fig" rid="F3">Fig. 3a</xref>)</bold>, and these interactions were also seen by IP of endogenous EXO1 <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 3a</xref>)</bold>. Exo1 bears two Cy motifs (at positions 204-206 and 293-295) <bold>(<xref ref-type="fig" rid="F1">Fig. 1d</xref>)</bold>. Mutation of both Cy motifs to Ala (CyMut) weakened interactions with CDK1 and particularly affected binding to CDK2 <bold>(<xref ref-type="fig" rid="F3">Fig. 3a</xref>)</bold>, as confirmed by reverse co-IP <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 3b</xref>)</bold>. Importantly, mutation of both Cy motifs dramatically reduced EXO1 phosphorylation, as assessed by IP-Westerns with both CDK substrate antibodies and the newly generated site-specific anti-p-T732 antibody <bold>(<xref ref-type="fig" rid="F3">Fig. 3b</xref>)</bold>. Cy mutations also resulted in severe defects in RPA, BrdU/ssDNA, and Rad51 foci formation <bold>(<xref ref-type="fig" rid="F3">Fig. 3c-e</xref>; <xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 3c-f</xref>)</bold>. Strikingly, the resection and HR defects caused by Cy mutations could be rescued, at least partially, by phospho-mimic substitutions at the four C-terminal S/TP sites (CyMut/4D). Therefore, EXO1 interacts with CDK-cyclin complexes via its Cy motifs, and these interactions are required for EXO1 phosphorylation and DNA end resection; however, this requirement can be overridden by phospho-mimic substitutions on EXO1, unequivocally demonstrating that phosphorylation of EXO1 by CDKs is essential for resection in vivo.</p></sec><sec id="S6"><title>EXO1 phosphorylation promotes its recruitment to DSBs</title><p id="P10">Next, we asked whether EXO1 phosphorylation might promote its recruitment to the sites of DSBs. We carried out live-cell imaging of green fluorescent protein (GFP)-tagged EXO1 at DNA breaks induced focally by laser micro-irradiation, as described before<sup><xref rid="R20" ref-type="bibr">20</xref></sup>. We have previously shown that EXO1 recruitment to breaks is significantly augmented in G2 relative to G1<sup><xref rid="R20" ref-type="bibr">20</xref></sup>. Therefore, to exclude any cell cycle effects, all GFP-EXO1 recruitment studies were carried out in cells synchronized in G2 (<bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 4a</xref></bold>). The CDK inhibitor AZD5438 attenuated GFP-EXO1 accumulation indicating that CDK activity is important for damage-site localization of EXO1 <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 4b</xref>)</bold>. In accord with this observation, the 4A-EXO1 mutant showed reduced accumulation at breaks while accumulation of 4D-EXO1 was augmented relative to WT-EXO1 <bold>(<xref ref-type="fig" rid="F4">Fig. 4a</xref>)</bold>. In parallel experiments, we quantified the recruitment of GFPRPA to the sites of laser-induced DNA lesions in real time, as described previously<sup><xref rid="R20" ref-type="bibr">20</xref></sup>. We observed that GFP-RPA accumulation was attenuated in cells expressing 4A-EXO1 and augmented in cells expressing 4D-EXO1 <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 4c</xref>)</bold>, indicating reduced and increased resection, respectively. Thus, phosphorylation of EXO1 by CDKs promotes its accumulation at DNA breaks which positively influences the extent of resection.</p><p id="P11">EXO1 is reported to interact with other proteins that control resection, namely CtIP<sup><xref rid="R30" ref-type="bibr">30</xref></sup> and BLM <sup><xref rid="R9" ref-type="bibr">9</xref></sup>. We asked whether EXO1 phosphorylation might augment its recruitment to DSBs by facilitating some of these protein-protein interactions. We carried out co-IPs using extracts from HEK-293 cells synchronized in G2 and expressing WT-, 4A-, or 4D-EXO1 <bold>(<xref ref-type="fig" rid="F4">Fig. 4b</xref>)</bold>. We observed interactions of EXO1 with CtIP and BLM but these interactions were not affected by the 4A mutations. Interestingly, we found that EXO1 also interacted with BRCA1, a key regulator of HR<sup><xref rid="R31" ref-type="bibr">31</xref></sup>, and this interaction was reduced by the 4A mutations <bold>(<xref ref-type="fig" rid="F4">Fig. 4b</xref>)</bold>, as confirmed by reverse co-IP <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 4d</xref>)</bold>. Binding to BRCA1 was unaffected by irradiation of cells or by the presence of ethidium bromide <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 4e</xref>)</bold>; thus, it is likely that these interactions were direct and not mediated by DNA. Notably, the BRCA1-EXO1 interaction was cell cycle dependent, showing a marked increase in cells synchronized in G2 <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 4f</xref>)</bold>. BRCA1 binding was also seen upon IP of endogenous EXO1 <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 4g</xref>)</bold>. Co-IPs with single mutants of the four S/TP sites did not show greatly reduced BRCA1 binding, indicating that the interaction of EXO1 with BRCA1 involves more than one phospho-S/TP site <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 4h</xref>)</bold>.</p><p id="P12">In agreement with the observed interaction of EXO1 and BRCA1, we found that EXO1 rapidly co-localized with BRCA1 at sites of micro-laser-induced DNA lesions <bold>(<xref ref-type="fig" rid="F4">Fig. 4c</xref>)</bold>. BRCA1 has previously been shown to interact with phosphorylated CtIP and augment its localization at DNA breaks<sup><xref rid="R12" ref-type="bibr">12</xref>,<xref rid="R13" ref-type="bibr">13</xref>,<xref rid="R32" ref-type="bibr">32</xref>,<xref rid="R33" ref-type="bibr">33</xref></sup>. To see if BRCA1 might have a similar role in EXO1 recruitment, we generated U2OS cells with stable shRNA-mediated BRCA1 knockdown and carried out live-cell imaging of DsRed-tagged EXO1 at DSBs induced by laser micro-irradiation, as described previously<sup><xref rid="R20" ref-type="bibr">20</xref></sup><bold>(<xref ref-type="fig" rid="F4">Fig. 4d</xref>)</bold>. We found that EXO1 recruitment was reduced in BRCA1-depleted cells, indicating a partial dependency on BRCA1 for damage-site localization. We reasoned that if phospho-EXO1 was dependent upon BRCA1 for recruitment to breaks, then 4D-EXO1 should not be able to complement the HR defect of BRCA1-deficient cells, even though it is capable of alleviating HR defects caused by EXO1 depletion <bold>(<xref ref-type="fig" rid="F2">Fig. 2c</xref>)</bold>, mutation of Cy sites on Exo1 <bold>(<xref ref-type="fig" rid="F3">Fig. 3e</xref>)</bold>, or pharmacological inhibition of CDKs <bold>(<xref ref-type="fig" rid="F2">Fig. 2f</xref>)</bold>. Indeed, we found that expression of 4DEXO1 could not correct defective Rad51 foci formation in BRCA1-depleted cells <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 4i</xref>)</bold> or reduce induction of radial chromosome structures<sup><xref rid="R34" ref-type="bibr">34</xref></sup> upon irradiation <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 4j</xref>)</bold>. In sum, although BRCA1 has a role in facilitating the recruitment of EXO1 to DSBs, whether the interactions between BRCA1 and EXO1 contribute to EXO1 recruitment remains to be determined.</p></sec><sec id="S7"><title>EXO1 phosphorylation regulates repair pathway choice</title><p id="P13">Our results raised the possibility that phosphorylation of EXO1 by CDKs is a novel mechanism by which the cell exercises repair pathway choice. To address this, we utilized two different reporter assays to quantify the repair of I-SceI-induced DSBs by HR<sup><xref rid="R17" ref-type="bibr">17</xref></sup> or by NHEJ<sup><xref rid="R35" ref-type="bibr">35</xref></sup>. Cells bearing integrated HR or NHEJ reporters were depleted of endogenous EXO1 and complemented with siRNA-resistant WT-, 4A-, or 4D-EXO1. Expression of 4A-EXO1 resulted in reduced levels of HR relative to WT-EXO1, while expression of 4D-EXO1 resulted in elevated levels of HR <bold>(<xref ref-type="fig" rid="F5">Fig. 5a</xref>)</bold>. Converse results were obtained with the NHEJ assay where expression of 4A-EXO1 resulted in increased levels of NHEJ relative to WT-EXO1 <bold>(<xref ref-type="fig" rid="F5">Fig. 5b</xref>)</bold>. The diametrically opposite effects of impaired EXO1 phosphorylation on HR and NHEJ clearly place this exonuclease at a pivotal point where the balance between these two repair pathways might be fine tuned by the cell.</p><p id="P14">Next, we irradiated U2OS cells expressing WT-, 4A-, or 4D-EXO1 and quantified the formation and dissolution of 53BP1 foci in G1 (Cyclin A negative) versus S/G2 (Cyclin A positive<sup><xref rid="R28" ref-type="bibr">28</xref></sup>) nuclei in order to evaluate the effects of phospho-mutant versus phospho-mimic EXO1 on the overall kinetics of DSB repair <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 5a</xref>)</bold>. Depletion of EXO1 had no discernible effect in G1 cells <bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 5b</xref>)</bold> but resulted in a subtle but distinct DSB repair defect in S/G2 cells, as seen by comparing sWT with rWT <bold>(<xref ref-type="fig" rid="F5">Fig. 5c</xref>)</bold>. This defect could be corrected by expression of 4D-EXO1 but not by expression of 4A-EXO1. Consequently, irradiated cells expressing 4A-EXO1 exhibited a striking induction of radial chromosomes upon IR <bold>(<xref ref-type="fig" rid="F5">Fig. 5d</xref>)</bold>, similar to that seen in BRCA1-deficient cells<sup><xref rid="R34" ref-type="bibr">34</xref></sup><bold>(<xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 4j</xref>)</bold>. Finally, cells expressing 4A-EXO1, but not those expressing 4DEXO1, were sensitive to IR in the G2 phase of the cell cycle <bold>(<xref ref-type="fig" rid="F5">Fig. 5e</xref>)</bold>. It is important to point out that while attenuated repair and compromised survival in 4A-EXO1-expressing cells are clearly attributable to defective HR, the extent to which the altered balance between NHEJ and HR contributes to these defects remains to be elucidated. Taken together, these results demonstrate that EXO1 phosphorylation is critical for DSB repair in S and G2 phases, maintenance of genomic integrity, and long-term cell survival in response to DNA damage.</p></sec></sec><sec sec-type="discussion" id="S8"><title>DISCUSSION</title><p id="P15">It is well established that CDKs regulate repair pathway choice by controlling the first step in resection (initiation) by phosphorylating CtIP at seven S/TP sites<sup><xref rid="R10" ref-type="bibr">10</xref>-<xref rid="R15" ref-type="bibr">15</xref></sup> and NBS1 at a single site<sup><xref rid="R16" ref-type="bibr">16</xref></sup>. The second step in resection (long-range resection) is indispensable for successful HR<sup><xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R4" ref-type="bibr">4</xref></sup>. Currently it is not known whether CDKs regulate this key step in human cells. Our results uncover a novel mechanism by which CDKs regulate the second step in resection by modifying EXO1, an exonuclease with critical functions in long-range resection.</p><p id="P16">EXO1 belongs to the RAD2/XPG family of nucleases and displays 5’-3’ exonuclease activity on single- and double-stranded DNA substrates, as well as flap structure-specific endonuclease activity. EXO1 plays important roles in DNA mismatch repair (MMR), mitotic and meiotic recombination, replication, and telomere homeostasis<sup><xref rid="R36" ref-type="bibr">36</xref></sup>. EXO1 is phosphorylated at S714 by ATM in response to DSBs<sup><xref rid="R17" ref-type="bibr">17</xref></sup> and by ATR in response to replication stress<sup><xref rid="R26" ref-type="bibr">26</xref></sup>, which influences its activity and stability, respectively. The N-terminal 300 amino acids of EXO1 encompassing its nuclease domain are highly conserved across species and share common sequences with other RAD2 nucleases<sup><xref rid="R24" ref-type="bibr">24</xref></sup>. In contrast, the C-terminal region of EXO1 is divergent and mediates interactions with the 14-3-3 checkpoint regulatory proteins<sup><xref rid="R37" ref-type="bibr">37</xref></sup>, the proliferating cell nuclear antigen<sup><xref rid="R38" ref-type="bibr">38</xref></sup>, and MMR proteins such as MutSα<sup><xref rid="R36" ref-type="bibr">36</xref></sup>. In the context of MMR, it has been proposed that the C terminus forms an unstructured regulatory domain that interacts with the N-terminal catalytic domain resulting in autoinhibition of EXO1; interaction of the C-terminus with MutSα not only couples EXO1 to the MMR machinery but also relieves inhibition of the catalytic domain<sup><xref rid="R24" ref-type="bibr">24</xref></sup>. How EXO1 is regulated in the context of HR and whether its C terminus plays any role in such regulation is currently not well understood.</p><p id="P17">We find that the C-terminal domain of EXO1 is phosphorylated by CDKs 1 and 2 at S639, T732, S815, and T824 during S and G2 phases of the cell cycle. Phosphorylation of these four sites promotes DNA end resection and DSB repair by HR, thereby preserving chromosomal integrity and promoting survival upon DNA damage. Further bolstering the connection between CDKs and EXO1, we find that Cy-mutant EXO1 (defective in cyclin-CDK binding) phenocopies 4A-EXO1 (all four phosphorylation sites mutated) in terms of resection and HR defects. Our results indicate that phosphorylation of EXO1 promotes end resection, in part, by augmenting the localization of EXO1 to DNA breaks possibly via interactions with BRCA1. The BRCA1-EXO1 interaction is limited to S/G2 phases, and this might serve to restrict resection to the post-replicative phases of the cell cycle. As even individual mutations of these four phosphorylation sites result in pronounced resection defects, it is possible that, individually, these four sites have additional and non-redundant functions that are yet to be discovered. Such a situation exists in the regulation of CtIP by CDKs. CtIP is phosphorylated at multiple sites by CDKs which promote resection in distinct ways such as facilitating interactions with Nbs1 and phosphorylation by ATM<sup><xref rid="R14" ref-type="bibr">14</xref></sup>, promoting phosphorylation by ATR<sup><xref rid="R5" ref-type="bibr">5</xref>,<xref rid="R11" ref-type="bibr">11</xref>,<xref rid="R39" ref-type="bibr">39</xref></sup>, and augmenting damage site localization via interaction with BRCA1<sup><xref rid="R12" ref-type="bibr">12</xref>,<xref rid="R13" ref-type="bibr">13</xref>,<xref rid="R15" ref-type="bibr">15</xref>,<xref rid="R40" ref-type="bibr">40</xref></sup>. A key question for the future is to determine the extent to which this type of regulation applies to EXO1.</p><p id="P18">With regard to the recruitment of EXO1 to DNA breaks, BRCA1 could also promote this step indirectly by augmenting CtIP binding<sup><xref rid="R40" ref-type="bibr">40</xref></sup> or antagonizing 53BP1-RIF1 binding<sup><xref rid="R41" ref-type="bibr">41</xref></sup> to DNA ends. It is important to point out that combined mutation of all four CDK-phosphorylation sites or depletion of BRCA1 attenuates, but does not abolish, damage site localization of EXO1. Therefore, it is likely that BRCA1 binding is only one mechanism by which EXO1 is recruited to DNA breaks and other mechanism(s), perhaps involving interactions with other resection proteins, are yet to be elucidated. In this regard, yeast MRX and Sae2 have been reported to promote the binding of Exo1 to DNA ends in vitro<sup><xref rid="R42" ref-type="bibr">42</xref></sup>. We too find that EXO1 binds to CtIP and BLM in vivo, as reported by others<sup><xref rid="R30" ref-type="bibr">30</xref>,<xref rid="R9" ref-type="bibr">9</xref></sup>, though binding is not affected by the phospho-site mutations. Thus, it is plausible that additional protein-protein interactions could serve to stabilize EXO1 at DNA breaks independently of these four phospho-S/TP sites. It is interesting that single mutants of EXO1, unlike 4A-EXO1, show no or minimal loss of BRCA1 binding yet exhibit pronounced resection defects. Although we cannot rule out subtle but critical BRCA1-interaction defects of the single EXO1 mutants that would not be revealed by co-IPs, this observation supports the hypothesis that additional, non-redundant mechanisms contribute to the resection defects of individual EXO1 mutants. Elucidating additional functions for the identified phosphorylation sites on EXO1 is an important task for the future and our study sets the stage for investigating such mechanisms. Finally, our results demonstrating regulation of long-range resection by CDKs in human cells have some precedence in yeast where Cdk1-dependent Dna2 phosphorylation has been shown to regulate DNA end resection<sup><xref rid="R43" ref-type="bibr">43</xref></sup>.</p><p id="P19">In sum, our findings indicate that phosphorylation of EXO1 by CDKs in S/G2 phases stimulates resection, in part, by promoting EXO1 accumulation at DSBs, and regulation of long-range resection in this manner is critical for repair pathway choice, maintenance of genomic integrity, and long-term survival in the face of genomic insults.</p></sec><sec sec-type="methods" id="S9"><title>METHODS</title><sec id="S10"><title>Cell culture and CDK inhibitor treatments</title><p id="P20">U2OS and HEK-293 cells (obtained from ATCC) were maintained in α-MEM and DMEM medium, respectively, supplemented with 10% fetal bovine serum and penicillin/streptomycin in a humidified atmosphere with 5% CO<sub>2</sub>. A U2OS line with stable shRNA-mediated knockdown of BRCA1 was generated by transfection with a pGIPZ vector (TATGTGGTCACACTTTGTG; No: V2LHS_254648, Thermo Scientific) using Lipofectamine2000 (Invitrogen) followed by selection and continued maintenance in puromycin (1 μg/ml, Invitrogen). Control cells were generated by transfection with a pGIPZ vector expressing scrambled shRNA (No: RHS4346, Thermo Scientific). For CDK inhibition, cells were incubated with 2μM AZD5438 (Selleck) or 10μM Roscovitine (Sigma) for 5 hours before cells were harvested or irradiated.</p></sec><sec id="S11"><title>Cell cycle synchronization and flow cytometry</title><p id="P21">HEK-293 cells were synchronized in G1 by treatment with aphidicolin (2μg/ml, Sigma) for 16 hours and then released into regular media for either 5 hours (S phase) or 10 hours (G2 phase)<sup><xref rid="R19" ref-type="bibr">19</xref></sup>. Alternatively, HEK-293 cells were synchronized in late G2 by treatment with nocodazole (100ng/ml, Sigma) for 16 hours. Cell cycle stage was determined by flow cytometry using a BD CYTOMICS FC500 Flow Cytometer (Becton, Dickinson and Company).</p></sec><sec id="S12"><title>Cloning and mutagenesis</title><p id="P22">EXO1b was subcloned from Flag2-EXO1<sup><xref rid="R17" ref-type="bibr">17</xref></sup> into pDONR221 (Invitrogen) using the Gateway® BP Clonase® II enzyme mix (Invitrogen). Site-directed mutagenesis of EXO1 was carried out using the QuikChange® II XL Site-Directed Mutagenesis kit (Stratagene); see <bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Table 1</xref></bold> for primer sequences. EXO1 was then transferred to pLenti6.3/V5-DEST (Invitrogen) for C-terminal V5-tagged fusion protein expression.</p></sec><sec id="S13"><title>Transfection of cells</title><p id="P23">Depletion of EXO1 or CDKs was carried out by transfection with appropriate siRNAs (<bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Table 2</xref></bold>; Invitrogen) using electroporation (Amaxa) for U2OS cells and Lipofectamine2000 (Invitrogen) for HEK-293 cells; cells were harvested 48 hours later to verify knockdown by Western blotting. Cells were transfected with scrambled siRNA (siScr) as controls. For expression of tagged proteins, cells were transfected with the appropriate expression plasmid and harvested after 24-48 hours. For ectopic expression of EXO1 in cells with knockdown of endogenous EXO1 or CDKs, cells were transfected with the expression plasmid 24 hours after siRNA transfection.</p></sec><sec id="S14"><title>Western blotting and antibodies</title><p id="P24">Nuclear extracts for Western blotting were prepared by re-suspending cell pellets in hypotonic lysis buffer (10mM Tris-HCl pH7.5, 1.5mM MgCl<sub>2</sub>, 5mM KCl, protease and phosphatase inhibitors), followed by nuclear extraction buffer (50mM Tris-HCl pH7.5, 0.5M NaCl, 2mM EDTA, 10% Sucrose, 10% Glycerol, protease and phosphatase inhibitors)<sup><xref rid="R19" ref-type="bibr">19</xref></sup>.The following primary antibodies were used: V5 [Invitrogen], Phospho-(Thr) CDK substrate, Phospho-(Ser) CDK substrate, CHK1, phospho-CHK1 (S317), phospho-CHK2 (T68) [Cell signaling], CDK1 (C-19), CDK2 (H-298, D-12), Rad51 (H-92), Cyclin B1 (H-433), 53BP1 (H-300) [Santa Cruz], actin [Sigma], RPA [Calbiochem], BrdU [Becton Dickinson], Cyclin A (6E6), GFP [Abcam], BRCA1, EXO1 monoclonal [ThermoFisher], EXO1 polyclonal, CtIP, BLM, CHK2 [Bethyl], DsRed [Clontech], and Ku80 [a kind gift from Dr. B.P. Chen]. The following secondary antibodies were used: HRP-conjugated secondary antibodies [Biorad], and Alexa488/568/647-conjugated secondary antibodies [Invitrogen]. Antibody dilutions and catalogue numbers are provided in <bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Table 3</xref></bold>. Rabbit polyclonal phospho-specific antibody against p-T732 of EXO1 was generated by Abmart using the phospho-peptide NH<sub>2</sub>-Cys-FSKKD(pT)PLRNK-COOH, where pT indicates phospho-Thr. Uncropped scans of Western blots are shown in <bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 6</xref></bold>.</p></sec><sec id="S15"><title>Immunoprecipitation</title><p id="P25">Whole cell extracts were prepared in lysis buffer (20mM Tris-HCl pH7.5, 80mM NaCl, 2mM EDTA, 10% Glycerol, 0.2% NP-40) with additional protease and phosphatase inhibitors. Antibodies were bound to Dynabeads (Invitrogen) and beads were incubated overnight with cell extracts, following manufacturer's protocol. Beads were washed extensively in lysis buffer and then boiled in 1X SDS loading buffer prior to Western blotting. Normal mouse IgG (Santa Cruz) was used for control immunoprecipitations.</p></sec><sec id="S16"><title>Irradiation of cells</title><p id="P26">Cells were irradiated with gamma rays from a cesium source (JL Shepherd and Associates). For laser irradiation, cells were pre-sensitized by overnight incubation with BrdU (10 μg/ml) and then micro-irradiated with a pulsed nitrogen laser (Spectra-Physics; 365nm, 10Hz) with output set at 70% of the maximum<sup><xref rid="R20" ref-type="bibr">20</xref></sup>. Cells were maintained at 37°C in 35 mm glass-bottom culture dishes (MatTek Cultureware) during micro-irradiation and image acquisition; the growth medium was replaced by CO<sub>2</sub>-independent medium (Invitrogen) before irradiation.</p></sec><sec id="S17"><title>Immunofluorescence staining</title><p id="P27">Cells were seeded onto glass chamber slides (Lab-Tek) and immunostained with anti-RPA antibody or co-immunostained with anti-Rad51 and anti-Cyclin A antibodies 3 hours after irradiation with 6 Gy. Cells were fixed with 4% PFA/PBS and permeabilized with 0.5% Triton X-100 before incubation with antibodies<sup><xref rid="R19" ref-type="bibr">19</xref></sup>. In order to obtain clear RPA foci, cells were subject to in situ fractionation<sup><xref rid="R44" ref-type="bibr">44</xref></sup>. The average number of RPA foci per nucleus or mean Rad51 foci for Cyclin A positive (S/G2) nuclei<sup><xref rid="R28" ref-type="bibr">28</xref></sup> was determined after scoring at least 50 nuclei and subtracting background (average numbers of foci in mock-irradiated cells). For quantifying DSB repair kinetics, cells were co-immunostained with anti-53BP1 and anti-Cyclin A antibodies at different time points post-irradiation (1 Gy), as described previously<sup><xref rid="R35" ref-type="bibr">35</xref></sup>. The average numbers of 53BP1 foci for Cyclin A positive (S/G2) and Cyclin A negative (G1) nuclei were determined after scoring at least 50 nuclei and subtracting background. Images were captured using a Leica DH5500B fluorescence microscope (40X objective lens) coupled to a Leica DFC340 FX camera using Leica Application Suite v3 acquisition software.</p></sec><sec id="S18"><title>BrdU/ssDNA assay</title><p id="P28">Cells grown in the presence of 10 μM BrdU (Sigma) for 16 hours were irradiated with 10 Gy of gamma rays and fixed one hour after irradiation. Cells were immunofluorescence stained with anti-BrdU antibody under non-denaturing conditions (in order to detect BrdU incorporated into ssDNA)<sup><xref rid="R20" ref-type="bibr">20</xref></sup>. For clarifying BrdU/ssDNA foci, cells were subject to in situ fractionation<sup><xref rid="R44" ref-type="bibr">44</xref></sup>. The percentage of BrdU positive cells (cells with 10 or more foci) was determined after scoring at least 50 nuclei and subtracting background.</p></sec><sec id="S19"><title>HR and NHEJ assays</title><p id="P29">To measure HR, GFP expression in MCF7 cells with an integrated DR-GFP reporter was quantified by flow cytometry<sup><xref rid="R17" ref-type="bibr">17</xref></sup>. To measure NHEJ, RFP expression in HEK-293 cells with an integrated GFP-to-RFP reporter was quantified by flow cytometry. For both assays, cells were co-transfected with siRNA against endogenous EXO1 and the V5-EXO1 expression plasmid; after 24 hours, cells were transfected with an <italic>I-SceI</italic> plasmid, and GFP or RFP expression was quantified by flow cytometry after an additional 72 hours. GFP+ or RFP+ frequencies were corrected for transfection efficiencies (measured simultaneously by parallel transfection with a wild type GFP expression vector).</p></sec><sec id="S20"><title>Metaphase chromosome preparations</title><p id="P30">Cells were irradiated with 6 Gy of gamma rays. Colcemid (Sigma), along with 1 mM caffeine (Sigma) to bypass G2/M arrest, was added at 8 hours post-IR. Metaphase chromosome spreads were prepared after 16 hours and scored for triradial and quadriradial chromosomes<sup><xref rid="R45" ref-type="bibr">45</xref></sup>.</p></sec><sec id="S21"><title>Live-cell imaging combined with laser micro-irradiation</title><p id="P31">Cells were transfected with GFP-EXO1, GFP-RPA, or DsRed-EXO1 constructs, laser micro-irradiated and time-lapse imaged, and the fluorescence intensities of micro-irradiated areas were plotted after background subtraction (fluorescence intensities of un-irradiated areas) <sup><xref rid="R20" ref-type="bibr">20</xref>,<xref rid="R46" ref-type="bibr">46</xref></sup>. Briefly, cells were irradiated and live-cell images were taken using a pulsed nitrogen laser (Spectra-Physics; 365nm, 10Hz) coupled to a Carl Zeiss Axiovert 200M microscope (63X oil-immersion objective). Fluorescence intensities were determined using Axiovision software v4.8 which converts signal intensities of accumulated GFP or DsRed into numerical values (arbitrary units). The fluorescence intensity of an un-irradiated region was subtracted from the fluorescence intensity of the micro-irradiated area for each nucleus at each time point in order to compensate for nonspecific fluorescence bleaching during repeated image acquisition. Mean value of the fluorescence intensities for each time point was calculated from at least 40 independent measurements. Total increase in fluorescence signal (after background subtraction) was plotted versus time.</p></sec><sec id="S22"><title>Colony formation assays</title><p id="P32">Cells were synchronized in G2 and irradiated with the indicated doses of gamma rays. After 4 hours, cells were plated in triplicate onto 60 mm dishes (1000 cells per dish). Surviving colonies were stained with crystal violet approximately 10-14 days later.</p></sec></sec><sec sec-type="supplementary-material" id="SM"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="SD1"><label>1</label><media xlink:href="NIHMS573163-supplement-1.pdf" orientation="portrait" xlink:type="simple" id="d37e1017" position="anchor"></media></supplementary-material></sec></body><back><ack id="S23"><title>ACKNOWLEDGEMENTS</title><p>SB is supported by grants from the National Institutes of Health (RO1 CA149461), National Aeronautics and Space Administration (NNX13AI13G), and the Cancer Prevention and Research Institute of Texas (RP100644). KK is supported by a National Health and Medical Research Council Senior Principal Research Fellowship. BL is supported by a grant from Foundation ARC. We are grateful to Prof. David Chen for facilitating the laser micro-irradiation experiments. MH and MI completed this work in partial fulfillment of the requirements for their PhD degrees.</p></ack><fn-group><fn id="FN2"><p id="P33">AUTHOR CONTRIBUTIONS</p><p id="P34">NT, BM, MP, BL, KK, and SB designed the experiments. NT, BM, MH, MI, CC, JH, and BL carried out the experiments and analyzed results. BM, BL, KKK, and SB wrote the paper.</p></fn><fn id="FN3"><p id="P35">CONFLICT OF INTEREST</p><p id="P36">The authors declare no conflict of interest.</p></fn></fn-group><ref-list><title>REFERENCES</title><ref id="R1"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wyman</surname><given-names>C</given-names></name><name><surname>Kanaar</surname><given-names>R</given-names></name></person-group><article-title>DNA double-strand break repair: all's well that ends well.</article-title><source>Annu Rev Genet</source><year>2006</year><volume>40</volume><fpage>363</fpage><lpage>383</lpage><pub-id pub-id-type="pmid">16895466</pub-id></element-citation></ref><ref id="R2"><label>2</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Malumbres</surname><given-names>M</given-names></name><name><surname>Barbacid</surname><given-names>M</given-names></name></person-group><article-title>Mammalian cyclin-dependent kinases.</article-title><source>Trends Biochem Sci</source><year>2005</year><volume>30</volume><fpage>630</fpage><lpage>641</lpage><pub-id pub-id-type="pmid">16236519</pub-id></element-citation></ref><ref id="R3"><label>3</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Symington</surname><given-names>LS</given-names></name><name><surname>Gautier</surname><given-names>J</given-names></name></person-group><article-title>Double-strand break end resection and repair pathway choice.</article-title><source>Annu Rev Genet</source><year>2011</year><volume>45</volume><fpage>247</fpage><lpage>271</lpage><pub-id pub-id-type="pmid">21910633</pub-id></element-citation></ref><ref id="R4"><label>4</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ferretti</surname><given-names>LP</given-names></name><name><surname>Lafranchi</surname><given-names>L</given-names></name><name><surname>Sartori</surname><given-names>AA</given-names></name></person-group><article-title>Controlling DNA-end resection: a new task for CDKs.</article-title><source>Front Genet</source><year>2013</year><volume>4</volume><fpage>99</fpage><pub-id pub-id-type="pmid">23760669</pub-id></element-citation></ref><ref id="R5"><label>5</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Huertas</surname><given-names>P</given-names></name></person-group><article-title>DNA resection in eukaryotes: deciding how to fix the break.</article-title><source>Nat Struct Mol Biol</source><year>2010</year><volume>17</volume><fpage>11</fpage><lpage>16</lpage><pub-id pub-id-type="pmid">20051983</pub-id></element-citation></ref><ref id="R6"><label>6</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gravel</surname><given-names>S</given-names></name><name><surname>Chapman</surname><given-names>JR</given-names></name><name><surname>Magill</surname><given-names>C</given-names></name><name><surname>Jackson</surname><given-names>SP</given-names></name></person-group><article-title>DNA helicases Sgs1 and BLM promote DNA double-strand break resection.</article-title><source>Genes Dev</source><year>2008</year><volume>22</volume><fpage>2767</fpage><lpage>2772</lpage><pub-id pub-id-type="pmid">18923075</pub-id></element-citation></ref><ref id="R7"><label>7</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mimitou</surname><given-names>EP</given-names></name><name><surname>Symington</surname><given-names>LS</given-names></name></person-group><article-title>Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing.</article-title><source>Nature</source><year>2008</year><volume>455</volume><fpage>770</fpage><lpage>774</lpage><pub-id pub-id-type="pmid">18806779</pub-id></element-citation></ref><ref id="R8"><label>8</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhu</surname><given-names>Z</given-names></name><name><surname>Chung</surname><given-names>WH</given-names></name><name><surname>Shim</surname><given-names>EY</given-names></name><name><surname>Lee</surname><given-names>SE</given-names></name><name><surname>Ira</surname><given-names>G</given-names></name></person-group><article-title>Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.</article-title><source>Cell</source><year>2008</year><volume>134</volume><fpage>981</fpage><lpage>994</lpage><pub-id pub-id-type="pmid">18805091</pub-id></element-citation></ref><ref id="R9"><label>9</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nimonkar</surname><given-names>AV</given-names></name><etal></etal></person-group><article-title>BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair.</article-title><source>Genes Dev</source><year>2011</year><volume>25</volume><fpage>350</fpage><lpage>362</lpage><pub-id pub-id-type="pmid">21325134</pub-id></element-citation></ref><ref id="R10"><label>10</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Huertas</surname><given-names>P</given-names></name><name><surname>Jackson</surname><given-names>SP</given-names></name></person-group><article-title>Human CtIP mediates cell cycle control of DNA end resection and double strand break repair.</article-title><source>J Biol Chem</source><year>2009</year><volume>284</volume><fpage>9558</fpage><lpage>9565</lpage><pub-id pub-id-type="pmid">19202191</pub-id></element-citation></ref><ref id="R11"><label>11</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Huertas</surname><given-names>P</given-names></name><name><surname>Cortes-Ledesma</surname><given-names>F</given-names></name><name><surname>Sartori</surname><given-names>AA</given-names></name><name><surname>Aguilera</surname><given-names>A</given-names></name><name><surname>Jackson</surname><given-names>SP</given-names></name></person-group><article-title>CDK targets Sae2 to control DNA-end resection and homologous recombination.</article-title><source>Nature</source><year>2008</year><volume>455</volume><fpage>689</fpage><lpage>692</lpage><pub-id pub-id-type="pmid">18716619</pub-id></element-citation></ref><ref id="R12"><label>12</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yun</surname><given-names>MH</given-names></name><name><surname>Hiom</surname><given-names>K</given-names></name></person-group><article-title>CtIP-BRCA1 modulates the choice of DNA double-strand-break repair pathway throughout the cell cycle.</article-title><source>Nature</source><year>2009</year><volume>459</volume><fpage>460</fpage><lpage>463</lpage><pub-id pub-id-type="pmid">19357644</pub-id></element-citation></ref><ref id="R13"><label>13</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>L</given-names></name><name><surname>Nievera</surname><given-names>CJ</given-names></name><name><surname>Lee</surname><given-names>AY</given-names></name><name><surname>Wu</surname><given-names>X</given-names></name></person-group><article-title>Cell cycle-dependent complex formation of BRCA1.CtIP.MRN is important for DNA double-strand break repair.</article-title><source>J Biol Chem</source><year>2008</year><volume>283</volume><fpage>7713</fpage><lpage>7720</lpage><pub-id pub-id-type="pmid">18171670</pub-id></element-citation></ref><ref id="R14"><label>14</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>H</given-names></name><etal></etal></person-group><article-title>The interaction of CtIP and Nbs1 connects CDK and ATM to regulate HR-mediated double-strand break repair.</article-title><source>PLoS Genet</source><year>2013</year><volume>9</volume><fpage>e1003277</fpage><pub-id pub-id-type="pmid">23468639</pub-id></element-citation></ref><ref id="R15"><label>15</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Buis</surname><given-names>J</given-names></name><name><surname>Stoneham</surname><given-names>T</given-names></name><name><surname>Spehalski</surname><given-names>E</given-names></name><name><surname>Ferguson</surname><given-names>DO</given-names></name></person-group><article-title>Mre11 regulates CtIP-dependent double-strand break repair by interaction with CDK2.</article-title><source>Nat Struct Mol Biol</source><year>2012</year><volume>19</volume><fpage>246</fpage><lpage>252</lpage><pub-id pub-id-type="pmid">22231403</pub-id></element-citation></ref><ref id="R16"><label>16</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Falck</surname><given-names>J</given-names></name><etal></etal></person-group><article-title>CDK targeting of NBS1 promotes DNA-end resection, replication restart and homologous recombination.</article-title><source>EMBO Rep</source><year>2012</year><volume>13</volume><fpage>561</fpage><lpage>568</lpage><pub-id pub-id-type="pmid">22565321</pub-id></element-citation></ref><ref id="R17"><label>17</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bolderson</surname><given-names>E</given-names></name><etal></etal></person-group><article-title>Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks.</article-title><source>Nucleic Acids Res</source><year>2010</year><volume>38</volume><fpage>1821</fpage><lpage>1831</lpage><pub-id pub-id-type="pmid">20019063</pub-id></element-citation></ref><ref id="R18"><label>18</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Costelloe</surname><given-names>T</given-names></name><etal></etal></person-group><article-title>The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection.</article-title><source>Nature</source><year>2012</year><volume>489</volume><fpage>581</fpage><lpage>584</lpage><pub-id pub-id-type="pmid">22960744</pub-id></element-citation></ref><ref id="R19"><label>19</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tomimatsu</surname><given-names>N</given-names></name><name><surname>Mukherjee</surname><given-names>B</given-names></name><name><surname>Burma</surname><given-names>S</given-names></name></person-group><article-title>Distinct roles of ATR and DNA-PKcs in triggering DNA damage responses in ATM-deficient cells.</article-title><source>EMBO Rep</source><year>2009</year><volume>10</volume><fpage>629</fpage><lpage>635</lpage><pub-id pub-id-type="pmid">19444312</pub-id></element-citation></ref><ref id="R20"><label>20</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tomimatsu</surname><given-names>N</given-names></name><etal></etal></person-group><article-title>Exo1 plays a major role in DNA end resection in humans and influences double-strand break repair and damage signaling decisions.</article-title><source>DNA Repair (Amst)</source><year>2012</year><volume>11</volume><fpage>441</fpage><lpage>448</lpage><pub-id pub-id-type="pmid">22326273</pub-id></element-citation></ref><ref id="R21"><label>21</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Byth</surname><given-names>KF</given-names></name><etal></etal></person-group><article-title>AZD5438, a potent oral inhibitor of cyclin-dependent kinases 1, 2, and 9, leads to pharmacodynamic changes and potent antitumor effects in human tumor xenografts.</article-title><source>Mol Cancer Ther</source><year>2009</year><volume>8</volume><fpage>1856</fpage><lpage>1866</lpage><pub-id pub-id-type="pmid">19509270</pub-id></element-citation></ref><ref id="R22"><label>22</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Meijer</surname><given-names>L</given-names></name><etal></etal></person-group><article-title>Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5.</article-title><source>Eur J Biochem</source><year>1997</year><volume>243</volume><fpage>527</fpage><lpage>536</lpage><pub-id pub-id-type="pmid">9030781</pub-id></element-citation></ref><ref id="R23"><label>23</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stevenson-Lindert</surname><given-names>LM</given-names></name><name><surname>Fowler</surname><given-names>P</given-names></name><name><surname>Lew</surname><given-names>J</given-names></name></person-group><article-title>Substrate specificity of CDK2-cyclin A. What is optimal?</article-title><source>J Biol Chem</source><year>2003</year><volume>278</volume><fpage>50956</fpage><lpage>50960</lpage><pub-id pub-id-type="pmid">14506259</pub-id></element-citation></ref><ref id="R24"><label>24</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Orans</surname><given-names>J</given-names></name><etal></etal></person-group><article-title>Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family.</article-title><source>Cell</source><year>2011</year><volume>145</volume><fpage>212</fpage><lpage>223</lpage><pub-id pub-id-type="pmid">21496642</pub-id></element-citation></ref><ref id="R25"><label>25</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>RQ</given-names></name><etal></etal></person-group><article-title>CDC25B mediates rapamycin-induced oncogenic responses in cancer cells.</article-title><source>Cancer Res</source><year>2009</year><volume>69</volume><fpage>2663</fpage><lpage>2668</lpage><pub-id pub-id-type="pmid">19276368</pub-id></element-citation></ref><ref id="R26"><label>26</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>El-Shemerly</surname><given-names>M</given-names></name><name><surname>Hess</surname><given-names>D</given-names></name><name><surname>Pyakurel</surname><given-names>AK</given-names></name><name><surname>Moselhy</surname><given-names>S</given-names></name><name><surname>Ferrari</surname><given-names>S</given-names></name></person-group><article-title>ATR-dependent pathways control hEXO1 stability in response to stalled forks.</article-title><source>Nucleic Acids Res</source><year>2008</year><volume>36</volume><fpage>511</fpage><lpage>519</lpage><pub-id pub-id-type="pmid">18048416</pub-id></element-citation></ref><ref id="R27"><label>27</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shiromizu</surname><given-names>T</given-names></name><etal></etal></person-group><article-title>Identification of Missing Proteins in the neXtProt Database and Unregistered Phosphopeptides in the PhosphoSitePlus Database As Part of the Chromosome-Centric Human Proteome Project.</article-title><source>J Proteome Res</source><year>2013</year><comment>doi:10.1021/pr300825v</comment></element-citation></ref><ref id="R28"><label>28</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bekker-Jensen</surname><given-names>S</given-names></name><etal></etal></person-group><article-title>Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks.</article-title><source>J Cell Biol</source><year>2006</year><volume>173</volume><fpage>195</fpage><lpage>206</lpage><pub-id pub-id-type="pmid">16618811</pub-id></element-citation></ref><ref id="R29"><label>29</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Takeda</surname><given-names>DY</given-names></name><name><surname>Wohlschlegel</surname><given-names>JA</given-names></name><name><surname>Dutta</surname><given-names>A</given-names></name></person-group><article-title>A bipartite substrate recognition motif for cyclin-dependent kinases.</article-title><source>J Biol Chem</source><year>2001</year><volume>276</volume><fpage>1993</fpage><lpage>1997</lpage><pub-id pub-id-type="pmid">11067844</pub-id></element-citation></ref><ref id="R30"><label>30</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Eid</surname><given-names>W</given-names></name><etal></etal></person-group><article-title>DNA end resection by CtIP and exonuclease 1 prevents genomic instability.</article-title><source>EMBO Rep</source><year>2010</year><volume>11</volume><fpage>962</fpage><lpage>968</lpage><pub-id pub-id-type="pmid">21052091</pub-id></element-citation></ref><ref id="R31"><label>31</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Huen</surname><given-names>MS</given-names></name><name><surname>Sy</surname><given-names>SM</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name></person-group><article-title>BRCA1 and its toolbox for the maintenance of genome integrity.</article-title><source>Nat Rev Mol Cell Biol</source><year>2010</year><volume>11</volume><fpage>138</fpage><lpage>148</lpage><pub-id pub-id-type="pmid">20029420</pub-id></element-citation></ref><ref id="R32"><label>32</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Greenberg</surname><given-names>RA</given-names></name><etal></etal></person-group><article-title>Multifactorial contributions to an acute DNA damage response by BRCA1/BARD1-containing complexes.</article-title><source>Genes Dev</source><year>2006</year><volume>20</volume><fpage>34</fpage><lpage>46</lpage><pub-id pub-id-type="pmid">16391231</pub-id></element-citation></ref><ref id="R33"><label>33</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yu</surname><given-names>X</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name></person-group><article-title>DNA damage-induced cell cycle checkpoint control requires CtIP, a phosphorylation-dependent binding partner of BRCA1 C-terminal domains.</article-title><source>Mol Cell Biol</source><year>2004</year><volume>24</volume><fpage>9478</fpage><lpage>9486</lpage><pub-id pub-id-type="pmid">15485915</pub-id></element-citation></ref><ref id="R34"><label>34</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bunting</surname><given-names>SF</given-names></name><etal></etal></person-group><article-title>53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks.</article-title><source>Cell</source><year>2010</year><volume>141</volume><fpage>243</fpage><lpage>254</lpage><pub-id pub-id-type="pmid">20362325</pub-id></element-citation></ref><ref id="R35"><label>35</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mukherjee</surname><given-names>B</given-names></name><etal></etal></person-group><article-title>The dual PI3K/mTOR inhibitor NVP-BEZ235 is a potent inhibitor of ATM- and DNA-PKCs-mediated DNA damage responses.</article-title><source>Neoplasia</source><year>2012</year><volume>14</volume><fpage>34</fpage><lpage>43</lpage><pub-id pub-id-type="pmid">22355272</pub-id></element-citation></ref><ref id="R36"><label>36</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tran</surname><given-names>PT</given-names></name><name><surname>Erdeniz</surname><given-names>N</given-names></name><name><surname>Symington</surname><given-names>LS</given-names></name><name><surname>Liskay</surname><given-names>RM</given-names></name></person-group><article-title>EXO1-A multi-tasking eukaryotic nuclease.</article-title><source>DNA Repair (Amst)</source><year>2004</year><volume>3</volume><fpage>1549</fpage><lpage>1559</lpage><pub-id pub-id-type="pmid">15474417</pub-id></element-citation></ref><ref id="R37"><label>37</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Andersen</surname><given-names>SD</given-names></name><etal></etal></person-group><article-title>14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif.</article-title><source>DNA Repair (Amst)</source><year>2012</year><volume>11</volume><fpage>267</fpage><lpage>277</lpage><pub-id pub-id-type="pmid">22222486</pub-id></element-citation></ref><ref id="R38"><label>38</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liberti</surname><given-names>SE</given-names></name><etal></etal></person-group><article-title>Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks.</article-title><source>DNA Repair (Amst)</source><year>2011</year><volume>10</volume><fpage>73</fpage><lpage>86</lpage><pub-id pub-id-type="pmid">20970388</pub-id></element-citation></ref><ref id="R39"><label>39</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Peterson</surname><given-names>SE</given-names></name><etal></etal></person-group><article-title>Activation of DSB processing requires phosphorylation of CtIP by ATR.</article-title><source>Mol Cell</source><year>2013</year><volume>49</volume><fpage>657</fpage><lpage>667</lpage><pub-id pub-id-type="pmid">23273981</pub-id></element-citation></ref><ref id="R40"><label>40</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yu</surname><given-names>X</given-names></name><name><surname>Fu</surname><given-names>S</given-names></name><name><surname>Lai</surname><given-names>M</given-names></name><name><surname>Baer</surname><given-names>R</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name></person-group><article-title>BRCA1 ubiquitinates its phosphorylation-dependent binding partner CtIP.</article-title><source>Genes Dev</source><year>2006</year><volume>20</volume><fpage>1721</fpage><lpage>1726</lpage><pub-id pub-id-type="pmid">16818604</pub-id></element-citation></ref><ref id="R41"><label>41</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Daley</surname><given-names>JM</given-names></name><name><surname>Sung</surname><given-names>P</given-names></name></person-group><article-title>RIF1 in DNA break repair pathway choice.</article-title><source>Mol Cell</source><year>2013</year><volume>49</volume><fpage>840</fpage><lpage>841</lpage><pub-id pub-id-type="pmid">23473603</pub-id></element-citation></ref><ref id="R42"><label>42</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nicolette</surname><given-names>ML</given-names></name><etal></etal></person-group><article-title>Mre11-Rad50-Xrs2 and Sae2 promote 5' strand resection of DNA double-strand breaks.</article-title><source>Nat Struct Mol Biol</source><year>2010</year><volume>17</volume><fpage>1478</fpage><lpage>1485</lpage><pub-id pub-id-type="pmid">21102445</pub-id></element-citation></ref><ref id="R43"><label>43</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>X</given-names></name><etal></etal></person-group><article-title>Cell cycle regulation of DNA double-strand break end resection by Cdk1-dependent Dna2 phosphorylation.</article-title><source>Nat Struct Mol Biol</source><year>2011</year><volume>18</volume><fpage>1015</fpage><lpage>1019</lpage><pub-id pub-id-type="pmid">21841787</pub-id></element-citation></ref><ref id="R44"><label>44</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cuadrado</surname><given-names>M</given-names></name><etal></etal></person-group><article-title>ATM regulates ATR chromatin loading in response to DNA double-strand breaks.</article-title><source>J Exp Med</source><year>2006</year><volume>203</volume><fpage>297</fpage><lpage>303</lpage><pub-id pub-id-type="pmid">16461339</pub-id></element-citation></ref><ref id="R45"><label>45</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McEllin</surname><given-names>B</given-names></name><etal></etal></person-group><article-title>PTEN loss compromises homologous recombination repair in astrocytes: implications for glioblastoma therapy with temozolomide or poly(ADP-ribose) polymerase inhibitors.</article-title><source>Cancer Res</source><year>2010</year><volume>70</volume><fpage>5457</fpage><lpage>5464</lpage><pub-id pub-id-type="pmid">20530668</pub-id></element-citation></ref><ref id="R46"><label>46</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Moyal</surname><given-names>L</given-names></name><etal></etal></person-group><article-title>Requirement of ATM-dependent monoubiquitylation of histone H2B for timely repair of DNA double-strand breaks.</article-title><source>Mol Cell</source><year>2011</year><volume>41</volume><fpage>529</fpage><lpage>542</lpage><pub-id pub-id-type="pmid">21362549</pub-id></element-citation></ref></ref-list></back><floats-group><fig id="F1" orientation="portrait" position="float"><label>Figure 1</label><caption><title>EXO1 is phosphorylated by CDKs 1 and 2 in S and G2 phases</title><p><bold>(a</bold>) HEK-293 cells expressing V5-EXO1 were synchronized in different phases of the cell cycle. EXO1 was immunoprecipitated (IP) with anti-V5 antibody and Western blotted with anti-phospho-Ser (p-SP) or anti-phospho-Thr (p-TP) CDK substrate antibodies, as indicated. “+AZD” indicates cells pre-treated with the CDK inhibitor AZD5438 prior to analysis. <bold>(b)</bold> HEK-293 cells expressing V5-EXO1, synchronized in G2, were pre-treated with CDK inhibitors, AZD5438 or Roscovitine, prior to analysis of EXO1 phosphorylation by IP-Western. <bold>(c)</bold> HEK-293 cells expressing V5-EXO1, with siRNA-mediated depletion of CDK1 and/or CDK2, were synchronized in G2 and EXO1 phosphorylation status determined by IP-Western. Scr denotes scrambled siRNA used as control. <bold>(d)</bold> Schematic of human EXO1 with asterisks indicating location of S/TP sites. Sites determined in this study to be important for resection are numbered. Locations of cyclin-binding (Cy) sites are indicated by solid triangles. N-terminal (N) and Internal (I) catalytic domains are indicated. <bold>(e)</bold> U2OS cells were depleted of endogenous EXO1 using siRNA and complemented with siRNA-resistant wild type EXO1 (rWT) or EXO1 individually mutated at S/TP sites, as indicated. Cells were screened for resection defects by quantifying IR-induced RPA foci. Average numbers of RPA foci per cell are plotted after subtracting background (average numbers of foci in mock-irradiated cells). Cells expressing siRNA-sensitive EXO1 (sWT) or siRNA-resistant nuclease dead EXO1 (ND) served as resection-defective controls (dashed line demarcates basal levels of RPA foci in these cells). Western blot shows comparable expression of EXO1 mutants and depletion of sWT-EXO1 by siRNA. <bold>(f)</bold> HEK-293 cells expressing WT-EXO1 or EXO1 with all four C-terminal S/TP sites mutated to Ala (4A-EXO1) were synchronized in G2, and EXO1 phosphorylation status determined by IP-Western. <bold>(g)</bold> HEK-293 cells expressing WT-EXO1 or EXO1 with a T732A point mutation were synchronized in G1 or G2, as indicated, and EXO1 phosphorylation at the T732 site was determined by IP-Western with anti-phospho-EXO1 (p-T732) antibody. All experiments were replicated three times. Error bars depict S.E.M. See also <bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 1</xref></bold>.</p></caption><graphic xlink:href="nihms-573163-f0001"></graphic></fig><fig id="F2" orientation="portrait" position="float"><label>Figure 2</label><caption><title>Phosphorylation of EXO1 by CDKs promotes DNA end resection</title><p><bold>(a)</bold> U2OS cells were depleted of endogenous EXO1 using siRNA and complemented with siRNA-sensitive wild type EXO1 (sWT) or siRNA-resistant wild type EXO1 (rWT), nonphosphorylatable mutant EXO1 (4A), or phospho-mimic EXO1 with all four C-terminal S/TP sites replaced with Asp (4D), as indicated. Representative images of mock-irradiated or irradiated (IR) cells immunostained with anti-RPA antibody (red) are shown. Nuclei were stained with DAPI (blue). Plot shows average numbers of RPA foci per cell after subtracting background (average numbers of foci in mock-irradiated cells). <bold>(b)</bold> Representative images of mock-irradiated or irradiated cells immunostained for BrdU/ssDNA foci (red) are shown. Plot shows percentages of cells with more than 10 BrdU foci per nucleus after background subtraction. <bold>(c)</bold> Representative images of irradiated cells co-immunofluorescence stained with anti-Cyclin A antibody (red) and anti-Rad51 antibody (green) are shown. Average numbers of Rad51 foci for Cyclin A positive (S/G2) nuclei are plotted after background subtraction. <bold>(d,e,f)</bold> Cells expressing rWT or 4D EXO1 were pre-treated with CDK inhibitors (AZD5438 or Roscovitine) or with DMSO as control, irradiated, and assayed for RPA, BrdU, or Rad51 foci, as indicated. Scale bars denote 10 μm for all images. All experiments were replicated three times. Error bars depict S.E.M. See also <bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 2</xref></bold>.</p></caption><graphic xlink:href="nihms-573163-f0002"></graphic></fig><fig id="F3" orientation="portrait" position="float"><label>Figure 3</label><caption><title>Interaction of EXO1 and CDKs is obligatory for DNA end resection</title><p><bold>(a)</bold> HEK-293 cells expressing V5-tagged wild type EXO1 (rWT) or EXO1 with Cy (cyclin-binding) sites mutated (CyMut) were synchronized in G2. Whole cell extracts were immunoprecipitated with anti-V5 antibody and immunoprecipitates were probed by Western blotting with the indicated antibodies. <bold>(b)</bold> HEK-293 cells expressing WT- or CyMut-EXO1 were synchronized in G2. EXO1 was immunoprecipitated (IP) with anti-V5 antibody and Western blotted with anti-phospho-Ser (p-SP) or anti-phospho-Thr (p-TP) CDK substrate antibodies or with the site-specific anti-phospho-EXO1 (p-T732) antibody, as indicated. <bold>(c,d,e)</bold> U2OS cells were depleted of endogenous EXO1 using siRNA and complemented with siRNA-sensitive wild type EXO1 (sWT) or siRNA-resistant wild type EXO1 (rWT), Cy-mutant EXO1 (CyMut), or Cy-mutant EXO1 with phospho-mimic substitutions (CyMut/4D), as indicated. Cells were irradiated and assayed for RPA, BrdU/ssDNA, or Rad51 foci, as indicated. All experiments were replicated three times. Error bars depict S.E.M. See also <bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 3</xref></bold>.</p></caption><graphic xlink:href="nihms-573163-f0003"></graphic></fig><fig id="F4" orientation="portrait" position="float"><label>Figure 4</label><caption><title>EXO1 phosphorylation promotes its recruitment to DSBs</title><p><bold>(a)</bold> U2OS cells expressing wild type (WT), phospho-mutant (4A), or phospho-mimic (4D) GFPEXO1 were synchronized in G2 before laser micro-irradiation. Time-lapse images of accumulation of GFP-EXO1 (green) at DSBs induced by laser micro-irradiation (<italic>arrows</italic>) are shown. Plot shows recruitment of wild type or mutant forms of GFP-EXO1 after DNA damage. Western blot shows comparable levels of expression of GFP-EXO1. <bold>(b)</bold> HEK-293 cells expressing V5-tagged rWT-, 4A-, or 4D-EXO1 were synchronized in G2. Whole cell extracts were immunoprecipitated with anti-V5 antibody and immunoprecipitates were probed by Western blotting with the indicated antibodies. <bold>(c)</bold> U2OS cells expressing GFP-EXO1 (green) were co-immunofluorescence stained with anti-53BP1 antibody (red) and anti-BRCA1 antibody (purple) 5 minutes after laser micro-irradiation. Nuclei were stained with DAPI (blue). <bold>(d)</bold> U2OS cells with stable knockdown of BRCA1 (shBRCA1) or control cells (shScr), both expressing DsRed-EXO1, were synchronized in G2 before laser micro-irradiation. Cells transfected with shRNA vectors express GFP (green). Time-lapse images of accumulation of DsRed-EXO1 (red) at DSBs induced by laser micro-irradiation (<italic>arrows</italic>) are shown. Plot shows recruitment of DsRed-EXO1 after DNA damage. Western blot shows knockdown of BRCA1 and comparable levels of expression of DsRed-EXO1. Scale bars denote 10 μm for all images. All experiments were replicated three times. Error bars depict S.E.M. See also <bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 4</xref></bold>.</p></caption><graphic xlink:href="nihms-573163-f0004"></graphic></fig><fig id="F5" orientation="portrait" position="float"><label>Figure 5</label><caption><title>EXO1 phosphorylation regulates repair pathway choice</title><p><bold>(a)</bold> Homologous recombination efficiency was measured by quantifying GFP expression (by flow cytometry) in MCF7 cells harboring a DR-GFP reporter after transfection with an <italic>I-SceI</italic> plasmid. Cells were depleted of endogenous EXO1 using siRNA and complemented with sWT-, rWT-, 4A-, or 4D-EXO1, as indicated. Plot shows HR-directed repair relative to rWT-EXO1-expressing cells. Western blot shows comparable levels of expression of siRNA-resistant EXO1. <bold>(b)</bold> Non-homologous end joining efficiency was measured by quantifying RFP expression in HEK-293 cells harboring a GFP/RFP reporter after transfection with an <italic>I-SceI</italic> plasmid. The experiment was performed and data plotted as in (a). <bold>(c)</bold> U2OS cells depleted of endogenous EXO1 and expressing sWT-, rWT-, 4A-, or 4D-EXO1 were irradiated and co-immunostained for 53BP1 foci and Cyclin A (to demarcate cells in S/G2). Rates of DSB repair in S/G2 cells were determined by scoring 53BP1 foci in Cyclin A-positive nuclei. Percent foci remaining was plotted against the indicated times post-IR. <bold>(d)</bold> Percentages of metaphase spreads with one or more radial chromosomes (arrow) are plotted for irradiated U2OS cells with knockdown of endogenous EXO1 and with ectopic expression of wild type or mutant EXO1, as verified by Western blotting. <bold>(e)</bold> Plot shows clonogenic survival of U2OS cells with knockdown of endogenous EXO1 and with ectopic expression of wild type or mutant EXO1, as verified by Western blotting. Cells were synchronized in G2 and irradiated with the indicated doses before plating for colony formation. Scale bar denotes 5 μm. All experiments were replicated three times. Error bars depict S.E.M. See also <bold><xref ref-type="supplementary-material" rid="SD1">Supplementary Fig. 5</xref></bold>.</p></caption><graphic xlink:href="nihms-573163-f0005"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Asie/explor/AustralieFrV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 002D868 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 002D868 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Asie
|area= AustralieFrV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé=
|texte=
}}
| This area was generated with Dilib version V0.6.33. |  |