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Article: Efficient repair of DNA double-strand breaks in malignant cells with structural instability

TitleEfficient repair of DNA double-strand breaks in malignant cells with structural instability
Authors
KeywordsChromosomal rearrangement
DNA double-strand break (DSB)
I-SceI
Ovarian cancer
Issue Date2010
PublisherElsevier BV. The Journal's web site is located at http://www.elsevier.com/locate/mut
Citation
Mutation Research - Fundamental And Molecular Mechanisms Of Mutagenesis, 2010, v. 683 n. 1-2, p. 115-122 How to Cite?
AbstractAberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1α) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1α promoter from the TK gene, or deletion of either the EF1α promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR.
Persistent Identifierhttp://hdl.handle.net/10722/92987
ISSN
2015 Impact Factor: 2.581
2015 SCImago Journal Rankings: 0.703
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
NIH, NCI
Funding Information:

We thank Tamas Varga, Ilan R. Kirsch and Michael Kuehl for suggestions and helpful advice, and Maria Jasin for the gift of the pCBASce expression vector. This work was supported by the Intramural Research Program of the NIH, NCI.

References

 

DC FieldValueLanguage
dc.contributor.authorCheng, Yen_HK
dc.contributor.authorZhang, Zen_HK
dc.contributor.authorKeenan, Ben_HK
dc.contributor.authorRoschke, AVen_HK
dc.contributor.authorNakahara, Ken_HK
dc.contributor.authorAplan, PDen_HK
dc.date.accessioned2010-09-22T05:33:02Z-
dc.date.available2010-09-22T05:33:02Z-
dc.date.issued2010en_HK
dc.identifier.citationMutation Research - Fundamental And Molecular Mechanisms Of Mutagenesis, 2010, v. 683 n. 1-2, p. 115-122en_HK
dc.identifier.issn0027-5107en_HK
dc.identifier.urihttp://hdl.handle.net/10722/92987-
dc.description.abstractAberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1α) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1α promoter from the TK gene, or deletion of either the EF1α promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR.en_HK
dc.languageeng-
dc.publisherElsevier BV. The Journal's web site is located at http://www.elsevier.com/locate/muten_HK
dc.relation.ispartofMutation Research - Fundamental and Molecular Mechanisms of Mutagenesisen_HK
dc.subjectChromosomal rearrangementen_HK
dc.subjectDNA double-strand break (DSB)en_HK
dc.subjectI-SceIen_HK
dc.subjectOvarian canceren_HK
dc.subject.meshAntineoplastic Agents, Phytogenic - pharmacology-
dc.subject.meshChromosome Aberrations-
dc.subject.meshDNA Breaks, Double-Stranded-
dc.subject.meshDNA Repair - genetics-
dc.subject.meshOvarian Neoplasms - genetics - pathology-
dc.titleEfficient repair of DNA double-strand breaks in malignant cells with structural instabilityen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=1386-1964&volume=683&issue=1-2&spage=115&epage=122&date=2010&atitle=Efficient+repair+of+DNA+double-strand+breaks+in+malignant+cells+with+structural+instability-
dc.identifier.emailCheng, Y:yuecheng@hku.hken_HK
dc.identifier.authorityCheng, Y=rp01320en_HK
dc.description.naturelink_to_OA_fulltext-
dc.identifier.doi10.1016/j.mrfmmm.2009.10.016en_HK
dc.identifier.pmid19909760-
dc.identifier.pmcidPMC2797445-
dc.identifier.scopuseid_2-s2.0-71849097672en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-71849097672&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume683en_HK
dc.identifier.issue1-2en_HK
dc.identifier.spage115en_HK
dc.identifier.epage122en_HK
dc.identifier.isiWOS:000273845500016-
dc.publisher.placeNetherlandsen_HK
dc.identifier.scopusauthoridCheng, Y=36131038300en_HK
dc.identifier.scopusauthoridZhang, Z=8511330100en_HK
dc.identifier.scopusauthoridKeenan, B=35770945200en_HK
dc.identifier.scopusauthoridRoschke, AV=6507003058en_HK
dc.identifier.scopusauthoridNakahara, K=7202641691en_HK
dc.identifier.scopusauthoridAplan, PD=35404812500en_HK
dc.identifier.citeulike6193277-

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