BRCA1 and CtIP Are Both Required to Recruit Dna2 at Double-Strand Breaks in Homologous Recombination.

Nguyen Ngoc Hoa,Tanya T Paull,Soo-Hyun Yang,Mayumi Hirakawa,Shunichi Takeda,Kenshi Komatsu,Junya Kobayashi,Masato Omura,Hiroyuki Sasanuma,Brendan D Price

doi:10.1371/journal.pone.0124495

Nguyen Ngoc Hoa, Tanya T Paull + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0124495

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Abstract

Homologous recombination plays a key role in the repair of double-strand breaks (DSBs), and thereby significantly contributes to cellular tolerance to radiotherapy and some chemotherapy. DSB repair by homologous recombination is initiated by 5’ to 3’ strand resection (DSB resection), with nucleases generating the 3’ single-strand DNA (3’ssDNA) at DSB sites. Genetic studies of Saccharomyces cerevisiae demonstrate a two-step DSB resection, wherein CtIP and Mre11 nucleases carry out short-range DSB resection followed by long-range DSB resection done by Dna2 and Exo1 nucleases. Recent studies indicate that CtIP contributes to DSB resection through its non-catalytic role but not as a nuclease. However, it remains elusive how CtIP contributes to DSB resection. To explore the non-catalytic role, we examined the dynamics of Dna2 by developing an immuno-cytochemical method to detect ionizing-radiation (IR)-induced Dna2-subnuclear-focus formation at DSB sites in chicken DT40 and human cell lines. Ionizing-radiation induced Dna2 foci only in wild-type cells, but not in Dna2 depleted cells, with the number of foci reaching its maximum at 30 minutes and being hardly detectable at 120 minutes after IR. Induced foci were detectable in cells in the G2 phase but not in the G1 phase. These observations suggest that Dna2 foci represent the recruitment of Dna2 to DSB sites for DSB resection. Importantly, the depletion of CtIP inhibited the recruitment of Dna2 to DSB sites in both human cells and chicken DT40 cells. Likewise, a defect in breast cancer 1 (BRCA1), which physically interacts with CtIP and contributes to DSB resection, also inhibited the recruitment of Dna2. Moreover, CtIP physically associates with Dna2, and the association is enhanced by IR. We conclude that BRCA1 and CtIP contribute to DSB resection by recruiting Dna2 to damage sites, thus ensuring the robust DSB resection necessary for efficient homologous recombination.

Highlights

double-strand breaks (DSBs) are the most dangerous type of DNA damage, as a single unrepaired DSB can trigger apoptosis
The following evidences suggest that Dna2 foci form at the DSBs that are subjected to Dna2mediated resection
Dna2 foci were undetectable in the G1 phase, where no DSB resection occurs (Fig 5C and 5E)

Summary

Introduction

DSBs are the most dangerous type of DNA damage, as a single unrepaired DSB can trigger apoptosis. HR, but not NHEJ, repairs DSBs induced by chemotherapeutic agents as well as those that occur during physiological replication [1, 2]. The resulting 3’-overhang is coated with a single-strand binding protein known as replication protein A (RPA). Polymerized Rad performs homology search and strand invasion into the intact homologous sequences. In Saccharomyces cerevisiae, DSB resection is carried out by four nucleases: Mre11-Rad50-Xrs (the MRX complex), Sae, Dna and Exo1 [5,6,7,8,9]. MRX and Sae cooperatively initiate HR by removing up to a few hundred nucleotides from the 5’ end of DSBs, resulting in short-range resection [5, 6]. Dna and Exo perform the further long-range resection of two or more kilobases

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