Using 3′-Bridging Phosphorothiolates To Isolate the Forward DNA Cleavage Reaction of Human Topoisomerase IIα
Identifieur interne : 002A15 ( Main/Exploration ); précédent : 002A14; suivant : 002A16Using 3′-Bridging Phosphorothiolates To Isolate the Forward DNA Cleavage Reaction of Human Topoisomerase IIα
Auteurs : Joseph E. Deweese [États-Unis] ; Alex B. Burgin [États-Unis] ; Neil Osheroff [États-Unis]Source :
- Biochemistry [ 0006-2960 ] ; 2008.
Abstract
The ability to cleave DNA is critical to the cellular and pharmacological functions of human type II topoisomerases. However, the low level of cleavage at equilibrium and the tight coupling of the cleavage and ligation reactions make it difficult to characterize the mechanism by which these enzymes cut DNA. Therefore, to establish a system that isolates topoisomerase II-mediated DNA scission from ligation, oligonucleotide substrates were developed that contained a 3′-bridging phosphorothiolate at the scissile bond. Scission of these substrates generates a 3′-terminal −SH moiety that is a poor nucleophile relative to the normal 3′-terminal −OH group. Consequently, topoisomerase II cannot efficiently ligate phosphorothiolate substrates once they are cleaved. The characteristics of topoisomerase IIα-mediated cleavage of phosphorothiolate oligonucleotides were identical to those seen with wild-type substrates, except that no ligation was observed. This unidirectional accumulation of cleavage complexes provided critical information regarding coordination of the protomer subunits of topoisomerase IIα and the mechanism of action of topoisomerase II poisons. Results indicate that the two enzyme subunits are partially coordinated and that cleavage at one scissile bond increases the degree of cleavage at the other. Furthermore, anticancer drugs such as etoposide and amsacrine that strongly inhibit topoisomerase II-mediated DNA ligation have little effect on the forward scission reaction. In contrast, abasic sites that increase levels of cleavage complexes without affecting ligation stimulate the forward rate of scission. Phosphorothiolate substrates provide significant advantages over traditional “suicide substrates” and should be valuable for future studies on DNA scission and the topoisomerase II−DNA cleavage complex.
Url:
DOI: 10.1021/bi702194x
Affiliations:
Links toward previous steps (curation, corpus...)
- to stream Istex, to step Corpus: 002780
- to stream Istex, to step Curation: 002780
- to stream Istex, to step Checkpoint: 000729
- to stream Main, to step Merge: 002A41
- to stream Main, to step Curation: 002A15
Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Using 3′-Bridging Phosphorothiolates To Isolate the Forward DNA Cleavage Reaction of Human Topoisomerase IIα</title><author><name sortKey="Deweese, Joseph E" sort="Deweese, Joseph E" uniqKey="Deweese J" first="Joseph E." last="Deweese">Joseph E. Deweese</name></author><author><name sortKey="Burgin, Alex B" sort="Burgin, Alex B" uniqKey="Burgin A" first="Alex B." last="Burgin">Alex B. Burgin</name></author><author><name sortKey="Osheroff, Neil" sort="Osheroff, Neil" uniqKey="Osheroff N" first="Neil" last="Osheroff">Neil Osheroff</name></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:8F3634E0CF4B8D6E6F3F00E37F59AB1EC8B9338D</idno><date when="2008" year="2008">2008</date><idno type="doi">10.1021/bi702194x</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-HB2JZNVC-R/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002780</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002780</idno><idno type="wicri:Area/Istex/Curation">002780</idno><idno type="wicri:Area/Istex/Checkpoint">000729</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Checkpoint">000729</idno><idno type="wicri:doubleKey">0006-2960:2008:Deweese J:using:bridging:phosphorothiolates</idno><idno type="wicri:Area/Main/Merge">002A41</idno><idno type="wicri:Area/Main/Curation">002A15</idno><idno type="wicri:Area/Main/Exploration">002A15</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">Using 3′-Bridging Phosphorothiolates To Isolate the Forward DNA Cleavage Reaction of Human Topoisomerase IIα<ref type="bib" target="#fn2"></ref></title><author><name sortKey="Deweese, Joseph E" sort="Deweese, Joseph E" uniqKey="Deweese J" first="Joseph E." last="Deweese">Joseph E. Deweese</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Washington (État)</region></placeName><wicri:cityArea>Departments of Biochemistry and Medicine (Hematology/Oncology), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, and deCODE biostructures, Bainbridge Island</wicri:cityArea></affiliation><affiliation></affiliation></author><author><name sortKey="Burgin, Alex B" sort="Burgin, Alex B" uniqKey="Burgin A" first="Alex B." last="Burgin">Alex B. Burgin</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Washington (État)</region></placeName><wicri:cityArea>Departments of Biochemistry and Medicine (Hematology/Oncology), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, and deCODE biostructures, Bainbridge Island</wicri:cityArea></affiliation><affiliation></affiliation></author><author><name sortKey="Osheroff, Neil" sort="Osheroff, Neil" uniqKey="Osheroff N" first="Neil" last="Osheroff">Neil Osheroff</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Washington (État)</region></placeName><wicri:cityArea>Departments of Biochemistry and Medicine (Hematology/Oncology), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, and deCODE biostructures, Bainbridge Island</wicri:cityArea></affiliation><affiliation></affiliation><affiliation></affiliation><affiliation wicri:level="1"><country wicri:rule="url">États-Unis</country></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2008</date><date type="published">2008</date><biblScope unit="vol">47</biblScope><biblScope unit="issue">13</biblScope><biblScope unit="page" from="4129">4129</biblScope><biblScope unit="page" to="4140">4140</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">The ability to cleave DNA is critical to the cellular and pharmacological functions of human type II topoisomerases. However, the low level of cleavage at equilibrium and the tight coupling of the cleavage and ligation reactions make it difficult to characterize the mechanism by which these enzymes cut DNA. Therefore, to establish a system that isolates topoisomerase II-mediated DNA scission from ligation, oligonucleotide substrates were developed that contained a 3′-bridging phosphorothiolate at the scissile bond. Scission of these substrates generates a 3′-terminal −SH moiety that is a poor nucleophile relative to the normal 3′-terminal −OH group. Consequently, topoisomerase II cannot efficiently ligate phosphorothiolate substrates once they are cleaved. The characteristics of topoisomerase IIα-mediated cleavage of phosphorothiolate oligonucleotides were identical to those seen with wild-type substrates, except that no ligation was observed. This unidirectional accumulation of cleavage complexes provided critical information regarding coordination of the protomer subunits of topoisomerase IIα and the mechanism of action of topoisomerase II poisons. Results indicate that the two enzyme subunits are partially coordinated and that cleavage at one scissile bond increases the degree of cleavage at the other. Furthermore, anticancer drugs such as etoposide and amsacrine that strongly inhibit topoisomerase II-mediated DNA ligation have little effect on the forward scission reaction. In contrast, abasic sites that increase levels of cleavage complexes without affecting ligation stimulate the forward rate of scission. Phosphorothiolate substrates provide significant advantages over traditional “suicide substrates” and should be valuable for future studies on DNA scission and the topoisomerase II−DNA cleavage complex.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Washington (État)</li></region></list><tree><country name="États-Unis"><region name="Washington (État)"><name sortKey="Deweese, Joseph E" sort="Deweese, Joseph E" uniqKey="Deweese J" first="Joseph E." last="Deweese">Joseph E. Deweese</name></region><name sortKey="Burgin, Alex B" sort="Burgin, Alex B" uniqKey="Burgin A" first="Alex B." last="Burgin">Alex B. Burgin</name><name sortKey="Osheroff, Neil" sort="Osheroff, Neil" uniqKey="Osheroff N" first="Neil" last="Osheroff">Neil Osheroff</name><name sortKey="Osheroff, Neil" sort="Osheroff, Neil" uniqKey="Osheroff N" first="Neil" last="Osheroff">Neil Osheroff</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Main/Exploration
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 002A15 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd -nk 002A15 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= MersV1
|flux= Main
|étape= Exploration
|type= RBID
|clé= ISTEX:8F3634E0CF4B8D6E6F3F00E37F59AB1EC8B9338D
|texte= Using 3′-Bridging Phosphorothiolates To Isolate the Forward DNA Cleavage Reaction of Human Topoisomerase IIα
}}
| This area was generated with Dilib version V0.6.33. |  |