Diastereomeric Recognition of 5’,8-cyclo-2’-Deoxyadenosine Lesions by Human Poly(ADP-ribose) Polymerase 1 in a Biomimetic Model
Identifieur interne : 000340 ( Pmc/Checkpoint ); précédent : 000339; suivant : 000341Diastereomeric Recognition of 5’,8-cyclo-2’-Deoxyadenosine Lesions by Human Poly(ADP-ribose) Polymerase 1 in a Biomimetic Model
Auteurs : Annalisa Masi ; Arianna Sabbia ; Carla Ferreri ; Francesco Manoli ; Yanhao Lai ; Eduardo Laverde ; Yuan Liu [États-Unis] ; Marios G. Krokidis ; Chryssostomos Chatgilialoglu ; Maria Rosaria Faraone MennellaSource :
- Cells [ 2073-4409 ] ; 2019.
Abstract
5’,8-Cyclo-2’-deoxyadenosine (cdA), in the 5’
Url:
DOI: 10.3390/cells8020116
PubMed: 30717407
PubMed Central: 6406461
Affiliations:
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PMC:6406461Le document en format XML
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<author><name sortKey="Masi, Annalisa" sort="Masi, Annalisa" uniqKey="Masi A" first="Annalisa" last="Masi">Annalisa Masi</name>
<affiliation><nlm:aff id="af1-cells-08-00116">Istituto per la Sintesi Organica e la Fotoreattività, Consiglio Nazionale delle Ricerche, 40129 Bologna, Italy;<email>francesco.manoli@isof.cnr.it</email>
(F.M.);<email>chrys@isof.cnr.it</email>
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<author><name sortKey="Sabbia, Arianna" sort="Sabbia, Arianna" uniqKey="Sabbia A" first="Arianna" last="Sabbia">Arianna Sabbia</name>
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(F.M.);<email>chrys@isof.cnr.it</email>
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(F.M.);<email>chrys@isof.cnr.it</email>
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<author><name sortKey="Lai, Yanhao" sort="Lai, Yanhao" uniqKey="Lai Y" first="Yanhao" last="Lai">Yanhao Lai</name>
<affiliation><nlm:aff id="af3-cells-08-00116">Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA;<email>yalai@fiu.edu</email>
(Y.L.);<email>yualiu@fiu.edu</email>
(Y.L.)</nlm:aff>
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<author><name sortKey="Laverde, Eduardo" sort="Laverde, Eduardo" uniqKey="Laverde E" first="Eduardo" last="Laverde">Eduardo Laverde</name>
<affiliation><nlm:aff id="af4-cells-08-00116">Biochemistry Ph.D. Program, Florida International University, Miami, FL 33199, USA;<email>elave006@fiu.edu</email>
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<author><name sortKey="Liu, Yuan" sort="Liu, Yuan" uniqKey="Liu Y" first="Yuan" last="Liu">Yuan Liu</name>
<affiliation><nlm:aff id="af3-cells-08-00116">Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA;<email>yalai@fiu.edu</email>
(Y.L.);<email>yualiu@fiu.edu</email>
(Y.L.)</nlm:aff>
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<author><name sortKey="Krokidis, Marios G" sort="Krokidis, Marios G" uniqKey="Krokidis M" first="Marios G." last="Krokidis">Marios G. Krokidis</name>
<affiliation><nlm:aff id="af6-cells-08-00116">Institute of Nanoscience and Nanotechnology, NCSR Demokritos Agia Paraskevi, 15310 Athens, Greece;<email>m.krokidis@inn.demokritos.gr</email>
</nlm:aff>
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<author><name sortKey="Chatgilialoglu, Chryssostomos" sort="Chatgilialoglu, Chryssostomos" uniqKey="Chatgilialoglu C" first="Chryssostomos" last="Chatgilialoglu">Chryssostomos Chatgilialoglu</name>
<affiliation><nlm:aff id="af1-cells-08-00116">Istituto per la Sintesi Organica e la Fotoreattività, Consiglio Nazionale delle Ricerche, 40129 Bologna, Italy;<email>francesco.manoli@isof.cnr.it</email>
(F.M.);<email>chrys@isof.cnr.it</email>
(C.C.)</nlm:aff>
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<author><name sortKey="Faraone Mennella, Maria Rosaria" sort="Faraone Mennella, Maria Rosaria" uniqKey="Faraone Mennella M" first="Maria Rosaria" last="Faraone Mennella">Maria Rosaria Faraone Mennella</name>
<affiliation><nlm:aff id="af2-cells-08-00116">Dipartimento di Biologia, Università di Napoli “Federico II”, 80138 Napoli, Italy;<email>ar.sabbia@studenti.unina.it</email>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Diastereomeric Recognition of 5’,8-cyclo-2’-Deoxyadenosine Lesions by Human Poly(ADP-ribose) Polymerase 1 in a Biomimetic Model</title>
<author><name sortKey="Masi, Annalisa" sort="Masi, Annalisa" uniqKey="Masi A" first="Annalisa" last="Masi">Annalisa Masi</name>
<affiliation><nlm:aff id="af1-cells-08-00116">Istituto per la Sintesi Organica e la Fotoreattività, Consiglio Nazionale delle Ricerche, 40129 Bologna, Italy;<email>francesco.manoli@isof.cnr.it</email>
(F.M.);<email>chrys@isof.cnr.it</email>
(C.C.)</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Sabbia, Arianna" sort="Sabbia, Arianna" uniqKey="Sabbia A" first="Arianna" last="Sabbia">Arianna Sabbia</name>
<affiliation><nlm:aff id="af2-cells-08-00116">Dipartimento di Biologia, Università di Napoli “Federico II”, 80138 Napoli, Italy;<email>ar.sabbia@studenti.unina.it</email>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ferreri, Carla" sort="Ferreri, Carla" uniqKey="Ferreri C" first="Carla" last="Ferreri">Carla Ferreri</name>
<affiliation><nlm:aff id="af1-cells-08-00116">Istituto per la Sintesi Organica e la Fotoreattività, Consiglio Nazionale delle Ricerche, 40129 Bologna, Italy;<email>francesco.manoli@isof.cnr.it</email>
(F.M.);<email>chrys@isof.cnr.it</email>
(C.C.)</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Manoli, Francesco" sort="Manoli, Francesco" uniqKey="Manoli F" first="Francesco" last="Manoli">Francesco Manoli</name>
<affiliation><nlm:aff id="af1-cells-08-00116">Istituto per la Sintesi Organica e la Fotoreattività, Consiglio Nazionale delle Ricerche, 40129 Bologna, Italy;<email>francesco.manoli@isof.cnr.it</email>
(F.M.);<email>chrys@isof.cnr.it</email>
(C.C.)</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Lai, Yanhao" sort="Lai, Yanhao" uniqKey="Lai Y" first="Yanhao" last="Lai">Yanhao Lai</name>
<affiliation><nlm:aff id="af3-cells-08-00116">Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA;<email>yalai@fiu.edu</email>
(Y.L.);<email>yualiu@fiu.edu</email>
(Y.L.)</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Laverde, Eduardo" sort="Laverde, Eduardo" uniqKey="Laverde E" first="Eduardo" last="Laverde">Eduardo Laverde</name>
<affiliation><nlm:aff id="af4-cells-08-00116">Biochemistry Ph.D. Program, Florida International University, Miami, FL 33199, USA;<email>elave006@fiu.edu</email>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Liu, Yuan" sort="Liu, Yuan" uniqKey="Liu Y" first="Yuan" last="Liu">Yuan Liu</name>
<affiliation><nlm:aff id="af3-cells-08-00116">Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA;<email>yalai@fiu.edu</email>
(Y.L.);<email>yualiu@fiu.edu</email>
(Y.L.)</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="af4-cells-08-00116">Biochemistry Ph.D. Program, Florida International University, Miami, FL 33199, USA;<email>elave006@fiu.edu</email>
</nlm:aff>
</affiliation>
<affiliation wicri:level="2"><nlm:aff id="af5-cells-08-00116">Biomolecular Sciences Institute, Florida International University, Miami, FL 33199, USA</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Biomolecular Sciences Institute, Florida International University, Miami, FL 33199</wicri:regionArea>
<placeName><region type="state">Floride</region>
</placeName>
</affiliation>
</author>
<author><name sortKey="Krokidis, Marios G" sort="Krokidis, Marios G" uniqKey="Krokidis M" first="Marios G." last="Krokidis">Marios G. Krokidis</name>
<affiliation><nlm:aff id="af6-cells-08-00116">Institute of Nanoscience and Nanotechnology, NCSR Demokritos Agia Paraskevi, 15310 Athens, Greece;<email>m.krokidis@inn.demokritos.gr</email>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Chatgilialoglu, Chryssostomos" sort="Chatgilialoglu, Chryssostomos" uniqKey="Chatgilialoglu C" first="Chryssostomos" last="Chatgilialoglu">Chryssostomos Chatgilialoglu</name>
<affiliation><nlm:aff id="af1-cells-08-00116">Istituto per la Sintesi Organica e la Fotoreattività, Consiglio Nazionale delle Ricerche, 40129 Bologna, Italy;<email>francesco.manoli@isof.cnr.it</email>
(F.M.);<email>chrys@isof.cnr.it</email>
(C.C.)</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Faraone Mennella, Maria Rosaria" sort="Faraone Mennella, Maria Rosaria" uniqKey="Faraone Mennella M" first="Maria Rosaria" last="Faraone Mennella">Maria Rosaria Faraone Mennella</name>
<affiliation><nlm:aff id="af2-cells-08-00116">Dipartimento di Biologia, Università di Napoli “Federico II”, 80138 Napoli, Italy;<email>ar.sabbia@studenti.unina.it</email>
</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">Cells</title>
<idno type="eISSN">2073-4409</idno>
<imprint><date when="2019">2019</date>
</imprint>
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<front><div type="abstract" xml:lang="en"><p>5’,8-Cyclo-2’-deoxyadenosine (cdA), in the 5’<italic>R</italic>
and 5’<italic>S</italic>
diastereomeric forms, are typical non strand-break oxidative DNA lesions, induced by hydroxyl radicals, with emerging importance as a molecular marker. These lesions are exclusively repaired by the nucleotide excision repair (NER) mechanism with a low efficiency, thus readily accumulating in the genome. Poly(ADP-ribose) polymerase1 (PARP1) acts as an early responder to DNA damage and plays a key role as a nick sensor in the maintenance of the integrity of the genome by recognizing nicked DNA. So far, it was unknown whether the two diastereomeric cdA lesions could induce specific PARP1 binding. Here, we provide the first evidence of PARP1 to selectively recognize the diastereomeric lesions of 5’<italic>S</italic>
-cdA and 5’<italic>R</italic>
-cdA in vitro as compared to deoxyadenosine in model DNA substrates (23-mers) by using circular dichroism, fluorescence spectroscopy, immunoblotting analysis, and gel mobility shift assay. Several features of the recognition of the damaged and undamaged oligonucleotides by PARP1 were characterized. Remarkably, PARP1 exhibits different affinities in binding to a double strand (ds) oligonucleotide, which incorporates cdA lesions in <italic>R</italic>
and <italic>S</italic>
diastereomeric form. In particular, PARP1 proved to bind oligonucleotides, including a 5’<italic>S</italic>
-cdA, with a higher affinity constant for the 5’<italic>S</italic>
lesion in a model of ds DNA than 5’<italic>R</italic>
-cdA, showing different recognition patterns, also compared with undamaged dA. This new finding highlights the ability of PARP1 to recognize and differentiate the distorted DNA backbone in a biomimetic system caused by different diastereomeric forms of a cdA lesion.</p>
</div>
</front>
<back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Chatgilialoglu, C" uniqKey="Chatgilialoglu C">C. Chatgilialoglu</name>
</author>
<author><name sortKey="Ferreri, C" uniqKey="Ferreri C">C. Ferreri</name>
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</analytic>
</biblStruct>
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<author><name sortKey="Brody, J R" uniqKey="Brody J">J.R. Brody</name>
</author>
<author><name sortKey="Armen, R S" uniqKey="Armen R">R.S. Armen</name>
</author>
<author><name sortKey="Pascal, J M" uniqKey="Pascal J">J.M. Pascal</name>
</author>
</analytic>
</biblStruct>
</listBibl>
</div1>
</back>
</TEI>
<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Cells</journal-id>
<journal-id journal-id-type="iso-abbrev">Cells</journal-id>
<journal-id journal-id-type="publisher-id">cells</journal-id>
<journal-title-group><journal-title>Cells</journal-title>
</journal-title-group>
<issn pub-type="epub">2073-4409</issn>
<publisher><publisher-name>MDPI</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">30717407</article-id>
<article-id pub-id-type="pmc">6406461</article-id>
<article-id pub-id-type="doi">10.3390/cells8020116</article-id>
<article-id pub-id-type="publisher-id">cells-08-00116</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Diastereomeric Recognition of 5’,8-cyclo-2’-Deoxyadenosine Lesions by Human Poly(ADP-ribose) Polymerase 1 in a Biomimetic Model</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Masi</surname>
<given-names>Annalisa</given-names>
</name>
<xref ref-type="aff" rid="af1-cells-08-00116">1</xref>
<xref rid="c1-cells-08-00116" ref-type="corresp">*</xref>
<xref ref-type="author-notes" rid="fn1-cells-08-00116">†</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Sabbia</surname>
<given-names>Arianna</given-names>
</name>
<xref ref-type="aff" rid="af2-cells-08-00116">2</xref>
</contrib>
<contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0002-1359-0869</contrib-id>
<name><surname>Ferreri</surname>
<given-names>Carla</given-names>
</name>
<xref ref-type="aff" rid="af1-cells-08-00116">1</xref>
<xref rid="c1-cells-08-00116" ref-type="corresp">*</xref>
<xref ref-type="author-notes" rid="fn1-cells-08-00116">†</xref>
</contrib>
<contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0003-2734-1625</contrib-id>
<name><surname>Manoli</surname>
<given-names>Francesco</given-names>
</name>
<xref ref-type="aff" rid="af1-cells-08-00116">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Lai</surname>
<given-names>Yanhao</given-names>
</name>
<xref ref-type="aff" rid="af3-cells-08-00116">3</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Laverde</surname>
<given-names>Eduardo</given-names>
</name>
<xref ref-type="aff" rid="af4-cells-08-00116">4</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Liu</surname>
<given-names>Yuan</given-names>
</name>
<xref ref-type="aff" rid="af3-cells-08-00116">3</xref>
<xref ref-type="aff" rid="af4-cells-08-00116">4</xref>
<xref ref-type="aff" rid="af5-cells-08-00116">5</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Krokidis</surname>
<given-names>Marios G.</given-names>
</name>
<xref ref-type="aff" rid="af6-cells-08-00116">6</xref>
</contrib>
<contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0003-2626-2925</contrib-id>
<name><surname>Chatgilialoglu</surname>
<given-names>Chryssostomos</given-names>
</name>
<xref ref-type="aff" rid="af1-cells-08-00116">1</xref>
</contrib>
<contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0003-1947-4700</contrib-id>
<name><surname>Faraone Mennella</surname>
<given-names>Maria Rosaria</given-names>
</name>
<xref ref-type="aff" rid="af2-cells-08-00116">2</xref>
<xref rid="c1-cells-08-00116" ref-type="corresp">*</xref>
<xref ref-type="author-notes" rid="fn1-cells-08-00116">†</xref>
</contrib>
</contrib-group>
<aff id="af1-cells-08-00116"><label>1</label>
Istituto per la Sintesi Organica e la Fotoreattività, Consiglio Nazionale delle Ricerche, 40129 Bologna, Italy;<email>francesco.manoli@isof.cnr.it</email>
(F.M.);<email>chrys@isof.cnr.it</email>
(C.C.)</aff>
<aff id="af2-cells-08-00116"><label>2</label>
Dipartimento di Biologia, Università di Napoli “Federico II”, 80138 Napoli, Italy;<email>ar.sabbia@studenti.unina.it</email>
</aff>
<aff id="af3-cells-08-00116"><label>3</label>
Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA;<email>yalai@fiu.edu</email>
(Y.L.);<email>yualiu@fiu.edu</email>
(Y.L.)</aff>
<aff id="af4-cells-08-00116"><label>4</label>
Biochemistry Ph.D. Program, Florida International University, Miami, FL 33199, USA;<email>elave006@fiu.edu</email>
</aff>
<aff id="af5-cells-08-00116"><label>5</label>
Biomolecular Sciences Institute, Florida International University, Miami, FL 33199, USA</aff>
<aff id="af6-cells-08-00116"><label>6</label>
Institute of Nanoscience and Nanotechnology, NCSR Demokritos Agia Paraskevi, 15310 Athens, Greece;<email>m.krokidis@inn.demokritos.gr</email>
</aff>
<author-notes><corresp id="c1-cells-08-00116"><label>*</label>
Correspondence: <email>annalisa.masi@isof.cnr.it</email>
(A.M.); <email>carla.ferreri@isof.cnr.it</email>
(C.F.); <email>faraone@unina.it</email>
(M.R.F.M.); Tel.: +39-051-6398313 (A.M.); +39-051-6398289 (C.F.); +39-081-679136 (M.R.F.M.)</corresp>
<fn id="fn1-cells-08-00116"><label>†</label>
<p>These authors contributed equally to the work.</p>
</fn>
</author-notes>
<pub-date pub-type="epub"><day>02</day>
<month>2</month>
<year>2019</year>
</pub-date>
<pub-date pub-type="collection"><month>2</month>
<year>2019</year>
</pub-date>
<volume>8</volume>
<issue>2</issue>
<elocation-id>116</elocation-id>
<history><date date-type="received"><day>21</day>
<month>12</month>
<year>2018</year>
</date>
<date date-type="accepted"><day>01</day>
<month>2</month>
<year>2019</year>
</date>
</history>
<permissions><copyright-statement>© 2019 by the authors.</copyright-statement>
<copyright-year>2019</copyright-year>
<license license-type="open-access"><license-p>Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>
).</license-p>
</license>
</permissions>
<abstract><p>5’,8-Cyclo-2’-deoxyadenosine (cdA), in the 5’<italic>R</italic>
and 5’<italic>S</italic>
diastereomeric forms, are typical non strand-break oxidative DNA lesions, induced by hydroxyl radicals, with emerging importance as a molecular marker. These lesions are exclusively repaired by the nucleotide excision repair (NER) mechanism with a low efficiency, thus readily accumulating in the genome. Poly(ADP-ribose) polymerase1 (PARP1) acts as an early responder to DNA damage and plays a key role as a nick sensor in the maintenance of the integrity of the genome by recognizing nicked DNA. So far, it was unknown whether the two diastereomeric cdA lesions could induce specific PARP1 binding. Here, we provide the first evidence of PARP1 to selectively recognize the diastereomeric lesions of 5’<italic>S</italic>
-cdA and 5’<italic>R</italic>
-cdA in vitro as compared to deoxyadenosine in model DNA substrates (23-mers) by using circular dichroism, fluorescence spectroscopy, immunoblotting analysis, and gel mobility shift assay. Several features of the recognition of the damaged and undamaged oligonucleotides by PARP1 were characterized. Remarkably, PARP1 exhibits different affinities in binding to a double strand (ds) oligonucleotide, which incorporates cdA lesions in <italic>R</italic>
and <italic>S</italic>
diastereomeric form. In particular, PARP1 proved to bind oligonucleotides, including a 5’<italic>S</italic>
-cdA, with a higher affinity constant for the 5’<italic>S</italic>
lesion in a model of ds DNA than 5’<italic>R</italic>
-cdA, showing different recognition patterns, also compared with undamaged dA. This new finding highlights the ability of PARP1 to recognize and differentiate the distorted DNA backbone in a biomimetic system caused by different diastereomeric forms of a cdA lesion.</p>
</abstract>
<kwd-group><kwd>human poly(ADP-ribose) polymerase 1 (PARP1)</kwd>
<kwd>PARP-DNA complex</kwd>
<kwd>DNA-protein binding</kwd>
<kwd>DNA repair</kwd>
<kwd>5’,8-Cyclopurine-2’-deoxynucleoside</kwd>
<kwd>DNA damage</kwd>
<kwd>DNA repair efficiency</kwd>
</kwd-group>
</article-meta>
</front>
<floats-group><fig id="cells-08-00116-f001" orientation="portrait" position="float"><label>Figure 1</label>
<caption><p>Structures and oligonucleotide sequences. (<bold>a</bold>
) Chemical structures of the diastereomeric purine 5’,8-cyclo-2’-deoxynucleosides. (<bold>b</bold>
) The sequences of the single stranded (ss) oligonucleotides used in this study. In the double stranded (ds) oligonucleotides, the template strand contains a ds-N (dA), or ds-5’<italic>S</italic>
or ds-5’<italic>R</italic>
and the complementary strand contains a T opposite to X.</p>
</caption>
<graphic xlink:href="cells-08-00116-g001"></graphic>
</fig>
<fig id="cells-08-00116-f002" orientation="portrait" position="float"><label>Figure 2</label>
<caption><p>Ultraviolet (UV)melting curves of 23-mer duplexes containing (<bold>a</bold>
) 5’<italic>S</italic>
-cdA lesion and (<bold>b</bold>
) 5’<italic>R</italic>
-cdA lesion. UV melting curve of unmodified double stranded is shown in <xref ref-type="app" rid="app1-cells-08-00116">Figure S3</xref>
.</p>
</caption>
<graphic xlink:href="cells-08-00116-g002"></graphic>
</fig>
<fig id="cells-08-00116-f003" orientation="portrait" position="float"><label>Figure 3</label>
<caption><p>Circular dichroism and immunoblotting analysis for determining PARP1 binding to ds-oligonucleotides with cdA lesions. (<bold>a</bold>
–<bold>c</bold>
) CD profiles of PARP1 without or with the substrates. Human PARP1 protein only (15 μg·mL<sup>−1</sup>
, black line), PARP1 along with 50 nM (<bold>a</bold>
), 100 nM (<bold>b</bold>
), or 200 nM (<bold>c</bold>
) ds-N (blue line), or ds-5’<italic>S</italic>
(green line) or ds-5’<italic>R</italic>
(red line), respectively. (<bold>d</bold>
–<bold>f</bold>
) Immunoblotting results of PARP1 in the presence of 50 nM (<bold>d</bold>
), 100 nM (<bold>e</bold>
), and 200 nM (<bold>f</bold>
) oligonucleotide substrate. Lanes 1, 5, and 9 correspond to PARP1 only. Lanes 2, 6, and 10 correspond to PARP1 with 50 nM, 100 nM, and 200 nM ds-N, respectively. Lanes 3, 7, and 11 correspond to PARP1 with 50 nM, 100 nM, and 200 nM substrates containing a ds-5’<italic>R</italic>
-cdA, respectively. Lanes 4, 8, and 12 correspond to PARP1 with 50 nM, 100 nM, and 200 nM substrates containing a ds-5’<italic>S</italic>
-cdA, respectively. The results in the panels d-f are from different immune blottings; the blots are shown in <xref ref-type="app" rid="app1-cells-08-00116">Figure S5</xref>
.</p>
</caption>
<graphic xlink:href="cells-08-00116-g003"></graphic>
</fig>
<fig id="cells-08-00116-f004" orientation="portrait" position="float"><label>Figure 4</label>
<caption><p>Gel mobility shift assay for determining PARP1 binding to ds-oligonucleotides with cdA lesions. Lanes 1, 5, and 9 correspond to the substrate only. Lanes 2, 6, and 10 correspond to the binding mixture with 30 nM PARP1. Lanes 3, 7, and 11 correspond to the binding mixture with 40 nM PARP1. Lanes 4, 8, and 12 correspond to the binding mixture with 50 nM PARP1. The DNA substrates are schematically illustrated above the gel. Grouping of gels are cropped from different parts of the same gel. The original gel is represented in <xref ref-type="app" rid="app1-cells-08-00116">Figure S6</xref>
. The gel mobility shift assay procedure is described in the Materials and Methods in the main text. * represents a phosphate group.</p>
</caption>
<graphic xlink:href="cells-08-00116-g004"></graphic>
</fig>
<fig id="cells-08-00116-f005" orientation="portrait" position="float"><label>Figure 5</label>
<caption><p>Intrinsic fluorescence of PARP1 in the absence and presence of DNA substrates. (<bold>a</bold>
–<bold>c</bold>
) The fluorescence profiles of PARP1 protein alone (15 μg·mL<sup>−1</sup>
, black line) or that of PARP1 along with 50 nM (<bold>a</bold>
), 100 nM (<bold>b</bold>
), and 200 nM (<bold>c</bold>
) of ds-N (blue line) or ds-5’<italic>S</italic>
(green line) or ds-5’<italic>R</italic>
(red line).</p>
</caption>
<graphic xlink:href="cells-08-00116-g005"></graphic>
</fig>
<table-wrap id="cells-08-00116-t001" orientation="portrait" position="float"><object-id pub-id-type="pii">cells-08-00116-t001_Table 1</object-id>
<label>Table 1</label>
<caption><p>Melting points, Tm, of 23-mer DNA duplexes.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">5′-d(GCA GAC ATA TCC TAG AGX CAT AT)-3′<break></break>
3′-d(CGT CTG TAT AGG ATC TCT GTA TA)-3′</th>
<th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Tm, °C</th>
</tr>
</thead>
<tbody><tr><td align="center" valign="middle" rowspan="1" colspan="1">X = dA (unmodified)</td>
<td align="center" valign="middle" rowspan="1" colspan="1">60.0 ± 0.3</td>
</tr>
<tr><td align="center" valign="middle" rowspan="1" colspan="1">X = 5’<italic>R</italic>
-cdA</td>
<td align="center" valign="middle" rowspan="1" colspan="1">59.0 ± 0.2</td>
</tr>
<tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">X = 5’<italic>S</italic>
-cdA</td>
<td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">58.0 ± 0.3</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap id="cells-08-00116-t002" orientation="portrait" position="float"><object-id pub-id-type="pii">cells-08-00116-t002_Table 2</object-id>
<label>Table 2</label>
<caption><p>Sequences and molecular masses of the synthesized ODNs.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Strands</th>
<th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Sequence (5’–3′) <sup>1</sup>
</th>
<th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Mass Calcd. (Da)</th>
<th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Mass Found <sup>2</sup>
(Da)</th>
</tr>
</thead>
<tbody><tr><td align="center" valign="middle" rowspan="1" colspan="1">ss-N</td>
<td align="center" valign="middle" rowspan="1" colspan="1">GCA GAC ATA TCC TAG AGA CAT AT</td>
<td align="center" valign="middle" rowspan="1" colspan="1">7040.7</td>
<td align="center" valign="middle" rowspan="1" colspan="1">7038.1</td>
</tr>
<tr><td align="center" valign="middle" rowspan="1" colspan="1">ss-5’<italic>S</italic>
</td>
<td align="center" valign="middle" rowspan="1" colspan="1">GCA GAC ATA TCC TAG AGX CAT AT</td>
<td align="center" valign="middle" rowspan="1" colspan="1">7038.7</td>
<td align="center" valign="middle" rowspan="1" colspan="1">7037.2</td>
</tr>
<tr><td align="center" valign="middle" rowspan="1" colspan="1">ss-5’<italic>R</italic>
</td>
<td align="center" valign="middle" rowspan="1" colspan="1">GCA GAC ATA TCC TAG AGX CAT AT</td>
<td align="center" valign="middle" rowspan="1" colspan="1">7038.7</td>
<td align="center" valign="middle" rowspan="1" colspan="1">7036.9</td>
</tr>
<tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">CS <sup>3</sup>
</td>
<td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">ATA TGT CTC TAG GAT ATG TCT GC</td>
<td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">7044.7</td>
<td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">7044.7</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn><p><sup>1</sup>
X is 5’<italic>S</italic>
-cdA for ss-5’S and 5’<italic>R</italic>
-cdA for ss-5’<italic>R</italic>
. <sup>2</sup>
All the oligonucleotide masses were obtained by MALDI-TOF in negative mode. [M − nH]<sup>−</sup>
. <sup>3</sup>
CS = Complementary strand. </p>
</fn>
</table-wrap-foot>
</table-wrap>
<table-wrap id="cells-08-00116-t003" orientation="portrait" position="float"><object-id pub-id-type="pii">cells-08-00116-t003_Table 3</object-id>
<label>Table 3</label>
<caption><p>Ellipticity values were converted into molar ellipticity Θ (deg·cm<sup>2</sup>
·nmol<sup>−1</sup>
) based on the molecular weight of PARP1 proteins, of human PARP1 alone and along with increasing concentrations (50 nM, 100 nM, 200 nM), of double stranded oligonucleotides (ds-N, ds-5’<italic>S</italic>
-cdA, ds-5’<italic>R</italic>
-cdA), respectively, at 220 nm. The results are illustrated as mean values of triplicate with errors ± 5%.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Compounds</th>
<th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Θ<sub>220nm</sub>
<break></break>
50 nM</th>
<th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Θ<sub>220nm</sub>
<break></break>
100 nM</th>
<th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Θ<sub>220nm</sub>
<break></break>
200 nM</th>
</tr>
</thead>
<tbody><tr><td align="center" valign="middle" rowspan="1" colspan="1">PARP1</td>
<td align="center" valign="middle" rowspan="1" colspan="1">−2.84</td>
<td align="center" valign="middle" rowspan="1" colspan="1">−2.84</td>
<td align="center" valign="middle" rowspan="1" colspan="1">−2.84</td>
</tr>
<tr><td align="center" valign="middle" rowspan="1" colspan="1">PARP1 + ds-N</td>
<td align="center" valign="middle" rowspan="1" colspan="1">−7.14</td>
<td align="center" valign="middle" rowspan="1" colspan="1">−7.79</td>
<td align="center" valign="middle" rowspan="1" colspan="1">−2.46</td>
</tr>
<tr><td align="center" valign="middle" rowspan="1" colspan="1">PARP1 + ds-5’<italic>S</italic>
</td>
<td align="center" valign="middle" rowspan="1" colspan="1">−7.96</td>
<td align="center" valign="middle" rowspan="1" colspan="1">−5.82</td>
<td align="center" valign="middle" rowspan="1" colspan="1">−0.53</td>
</tr>
<tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">PARP1 + ds-5’<italic>R</italic>
</td>
<td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">−9.09</td>
<td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">−8.26</td>
<td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">−7.26</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap id="cells-08-00116-t004" orientation="portrait" position="float"><object-id pub-id-type="pii">cells-08-00116-t004_Table 4</object-id>
<label>Table 4</label>
<caption><p>Affinity constants (<italic>K<sub>a</sub>
</italic>
) of PARP1 binding to substrates.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Substrates</th>
<th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Saturation at (nM)</th>
<th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1"><italic>K<sub>a</sub>
</italic>
* (M<sup>−1</sup>
)</th>
</tr>
</thead>
<tbody><tr><td align="center" valign="middle" rowspan="1" colspan="1">ds-N</td>
<td align="center" valign="middle" rowspan="1" colspan="1">100</td>
<td align="center" valign="middle" rowspan="1" colspan="1">1.51 × 10<sup>7</sup>
</td>
</tr>
<tr><td align="center" valign="middle" rowspan="1" colspan="1">ds-5’<italic>S</italic>
</td>
<td align="center" valign="middle" rowspan="1" colspan="1">200</td>
<td align="center" valign="middle" rowspan="1" colspan="1">3.19 × 10<sup>9</sup>
</td>
</tr>
<tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">ds-5’<italic>R</italic>
</td>
<td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">200</td>
<td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">1.21 × 10<sup>7</sup>
</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn><p>* Details of constant calculations at a saturating concentration of substrates are illustrated in Supporting Information (pages S7 and S8).</p>
</fn>
</table-wrap-foot>
</table-wrap>
</floats-group>
</pmc>
<affiliations><list><country><li>États-Unis</li>
</country>
<region><li>Floride</li>
</region>
</list>
<tree><noCountry><name sortKey="Chatgilialoglu, Chryssostomos" sort="Chatgilialoglu, Chryssostomos" uniqKey="Chatgilialoglu C" first="Chryssostomos" last="Chatgilialoglu">Chryssostomos Chatgilialoglu</name>
<name sortKey="Faraone Mennella, Maria Rosaria" sort="Faraone Mennella, Maria Rosaria" uniqKey="Faraone Mennella M" first="Maria Rosaria" last="Faraone Mennella">Maria Rosaria Faraone Mennella</name>
<name sortKey="Ferreri, Carla" sort="Ferreri, Carla" uniqKey="Ferreri C" first="Carla" last="Ferreri">Carla Ferreri</name>
<name sortKey="Krokidis, Marios G" sort="Krokidis, Marios G" uniqKey="Krokidis M" first="Marios G." last="Krokidis">Marios G. Krokidis</name>
<name sortKey="Lai, Yanhao" sort="Lai, Yanhao" uniqKey="Lai Y" first="Yanhao" last="Lai">Yanhao Lai</name>
<name sortKey="Laverde, Eduardo" sort="Laverde, Eduardo" uniqKey="Laverde E" first="Eduardo" last="Laverde">Eduardo Laverde</name>
<name sortKey="Manoli, Francesco" sort="Manoli, Francesco" uniqKey="Manoli F" first="Francesco" last="Manoli">Francesco Manoli</name>
<name sortKey="Masi, Annalisa" sort="Masi, Annalisa" uniqKey="Masi A" first="Annalisa" last="Masi">Annalisa Masi</name>
<name sortKey="Sabbia, Arianna" sort="Sabbia, Arianna" uniqKey="Sabbia A" first="Arianna" last="Sabbia">Arianna Sabbia</name>
</noCountry>
<country name="États-Unis"><region name="Floride"><name sortKey="Liu, Yuan" sort="Liu, Yuan" uniqKey="Liu Y" first="Yuan" last="Liu">Yuan Liu</name>
</region>
</country>
</tree>
</affiliations>
</record>
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