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PMID: 20107609 Published · epublish English Journal Article Research Support, Non-U.S. Gov't

Collaborative action of Brca1 and CtIP in elimination of covalent modifications from double-strand breaks to facilitate subsequent break repair.

PLoS genetics ·Vol. 6 ·No. 1 ·2010-01-22 ·Pages e1000828

Nakamura K, Kogame T, Oshiumi H, Shinohara A, Sumitomo Y, Agama K, Pommier Y, Tsutsui KM, Tsutsui K, Hartsuiker E, Ogi T, Takeda S, Taniguchi Y

Abstract

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3' and 5' ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3' single-strand overhang at "clean" DSBs, thus initiating homologous recombination (HR)-dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3' single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIP(S332A/-/-) cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP(+/-/-) cells. Finally, CtIP(S332A/-/-)BRCA1(-/-) and CtIP(+/-/-)BRCA1(-/-) showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.

MeSH Terms
Animals BRCA1 Protein/genetics,metabolism Cell Cycle Cell Line, Tumor Chickens DNA/genetics DNA Breaks, Double-Stranded DNA Repair Nuclear Proteins/genetics,metabolism Protein Binding Recombination, Genetic
Chemicals
BRCA1 Protein Nuclear Proteins DNA
Authors & Affiliations
13 authors, click to expand affiliations / ORCID
Nakamura Kyoko
Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Kogame Toshiaki
Oshiumi Hiroyuki
Shinohara Akira
Sumitomo Yoshiki
Agama Keli
Pommier Yves
Tsutsui Kimiko M
Tsutsui Ken
Hartsuiker Edgar
Ogi Tomoo
Takeda Shunichi
Taniguchi Yoshihito
Conflict of Interest

The authors have declared that no competing interests exist.

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Article Info
Journal
PLoS genetics
Abbr.
PLoS Genet
ISSN
1553-7404
DOI
10.1371/journal.pgen.1000828
Published
2010-01-22
Epub
2010-00-22
Pages
e1000828
Language
English
Region
United States
NLM ID
101239074
PMCID
PMC2809774
Subset
IM
Grants
Cancer Research UK · 11809 · United Kingdom
External Links
PubMed source DOI: 10.1371/journal.pgen.1000828 PMC Full Text View journal details
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