Repair of DNA strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.

To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ~170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of si...

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Autores principales:	Slawomir Kumala, Krzysztof Fujarewicz, Dheekollu Jayaraju, Joanna Rzeszowska-Wolny, Ronald Hancock
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b4ce648ea9c1414f86efa97f7ed2c567
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spelling	oai:doaj.org-article:b4ce648ea9c1414f86efa97f7ed2c5672021-11-18T07:59:37ZRepair of DNA strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.1932-620310.1371/journal.pone.0052966https://doaj.org/article/b4ce648ea9c1414f86efa97f7ed2c5672013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23382828/?tool=EBIhttps://doaj.org/toc/1932-6203To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ~170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20-30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair.Slawomir KumalaKrzysztof FujarewiczDheekollu JayarajuJoanna Rzeszowska-WolnyRonald HancockPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 1, p e52966 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Slawomir Kumala Krzysztof Fujarewicz Dheekollu Jayaraju Joanna Rzeszowska-Wolny Ronald Hancock Repair of DNA strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.
description	To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ~170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20-30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair.
format	article
author	Slawomir Kumala Krzysztof Fujarewicz Dheekollu Jayaraju Joanna Rzeszowska-Wolny Ronald Hancock
author_facet	Slawomir Kumala Krzysztof Fujarewicz Dheekollu Jayaraju Joanna Rzeszowska-Wolny Ronald Hancock
author_sort	Slawomir Kumala
title	Repair of DNA strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.
title_short	Repair of DNA strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.
title_full	Repair of DNA strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.
title_fullStr	Repair of DNA strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.
title_full_unstemmed	Repair of DNA strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.
title_sort	repair of dna strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/b4ce648ea9c1414f86efa97f7ed2c567
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Repair of DNA strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.

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