Biochemical mechanism of DSB end resection and its regulation

Daley, James M.; Niu, Hengyao; Miller, Adam S.; Sung, Patrick

doi:10.1016/j.dnarep.2015.04.015

Cited by 115 publications

(119 citation statements)

References 155 publications

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“…BRCC36 DUB Activity Limits DNA End Resection-DNA end resection generates 3Ј single-stranded DNA (ssDNA) overhangs and is an early and decisive process for committing DSB repair via the HR pathway (27)(28)(29). Given the requirement of BRCC36 DUB in restraining HR repair ( Fig.…”

Section: Resultsmentioning

confidence: 99%

The Lys63-deubiquitylating Enzyme BRCC36 Limits DNA Break Processing and Repair

Ng¹,

Wei

Lan

et al. 2016

Journal of Biological Chemistry

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Multisubunit protein assemblies offer integrated functionalities for efficient cell signal transduction control. One example of such protein assemblies, the BRCA1-A macromolecular complex, couples ubiquitin recognition and metabolism and promotes cellular responses to DNA damage. Specifically, the BRCA1-A complex not only recognizes Lys 63 -linked ubiquitin (K63-Ub) adducts at the damaged chromatin but is endowed with K63-Ub deubiquitylase (DUB) activity. To explore how the BRCA1-A DUB activity contributes to its function at DNA double strand breaks (DSBs), we used RNAi and genome editing approaches to target BRCC36, the protein subunit that confers the BRCA1-A complex its DUB activity. Intriguingly, we found that the K63-Ub DUB activity, although dispensable for maintaining the integrity of the macromolecular protein assembly, is important in enforcing the accumulation of the BRCA1-A complex onto DSBs. Inactivating BRCC36 DUB attenuated BRCA1-A functions at DSBs and led to unrestrained DSB end resection and hyperactive DNA repair. Together, our findings uncover a pivotal role of BRCC36 DUB in limiting DSB processing and repair and illustrate how cells may physically couple ubiquitin recognition and metabolizing activities for fine tuning of DNA repair processes. Active hydrolysis of ubiquitin polymers by deubiquitylases (DUBs)2 has emerged as important biochemical processes that underlie diverse signal transduction pathways, including cell responses to DNA damage (1). To date, around 94 human DUBs have been identified and are classified into five families: ubiquitin C-terminal hydrolases, ubiquitin-specific proteases, ovarian tumor proteases, Josephins, and JAB1/MPN/MOV4 metalloproteases. Notably, the JAB1/MPN/MOV4 metalloprotease family members are zinc-dependent metalloproteases, and the other families are cysteine proteases (1).BRCC36, originally reported as the BRCA1/BRCA2-containing complex subunit 36 (2), is endowed with DUB activity for lysine 63-linked ubiquitin polymers (K63-Ub). BRCC36 DUB relies on its zinc-dependent JAB1/MPN/MOV4 metalloprotease domain (3) and its interaction with Abraxas/KIAA0157 (3-5). Although BRCC36 forms distinct nuclear and cytoplasmic complexes (5, 6) and plays pleiotropic roles during cell proliferation, the K63-Ub DUB is arguably best known for its strong links with the breast and ovarian susceptible gene product BRCA1 in DNA damage responses (DDRs). Indeed, BRCC36 resides within the BRCA1-A macromolecular protein complex, which comprises the ubiquitin-binding protein RAP80, BRCC45/BRE, Abraxas/CCDC98, Merit40/NBA1, and BRCA1. The BRCA1-A complex is targeted to chromatin domains surrounding DNA double strand breaks (DSBs) by the ubiquitin-binding protein RAP80, which preferentially binds to K63-Ub adducts generated by the ubiquitin-conjugating enzyme UBC13 (3, 7-12). Abraxas plays a major scaffolding role to support the integrity of the BRCA1-A assembly and directly anchors the tumor suppressor BRCA1 via a phosphorylation-dependent interaction (13-15). Indeed, BRCA1, via it...

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Section: Resultsmentioning

confidence: 99%

The Lys63-deubiquitylating Enzyme BRCC36 Limits DNA Break Processing and Repair

Ng¹,

Wei

Lan

et al. 2016

Journal of Biological Chemistry

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“…Neither did we detect any DNA binding activity in Cdc24. 3 Besides Dna2 and RPA, there were no other replication-related proteins in the Cdc24 pulled-down fraction (Fig. 4A).…”

Section: Cdc24 Functions In Long-range End Resectionmentioning

confidence: 99%

“…UV, IR, and chemical agents such as camptothecin (CPT)) (1). If DSBs are left unrepaired or are repaired inappropriately, it will lead to a variety of mutations such as deletions, insertions, translocation, and genetic fusions, resulting in drastic genomic instability (2,3).…”

mentioning

confidence: 99%

Cdc24 Is Essential for Long-range End Resection in the Repair of Double-stranded DNA Breaks

Zhang¹,

Yu²,

Li³

et al. 2016

Journal of Biological Chemistry

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Edited by Patrick SungDouble-stranded DNA breaks (DSBs) are highly detrimental DNA lesions, which may be repaired by the homologous recombination-mediated repair pathway. The 5 to 3 direction of long-range end resection on one DNA strand, in which 3-single-stranded DNA overhangs are created from broken DNA ends, is an essential step in this pathway. Dna2 has been demonstrated as an essential nuclease in this event, but the molecular mechanism of how Dna2 is recruited to DNA break sites in vivo has not been elucidated. In this study, a novel recombination factor called Cdc24 was identified in fission yeast. We demonstrated that Cdc24 localizes to DNA break sites during the repair of DNA breaks and is an essential factor in long-range end resection. We also determined that Cdc24 plays a direct role in recruiting Dna2 to DNA break sites through its interaction with Dna2 and replication protein A (RPA). Further, this study revealed that RPA acts as the foundation for assembling the machinery for long-range end resection by its essential role in recruiting Cdc24 and Dna2 to DNA break sites. These results define Cdc24 as an essential factor for long-range end resection in the repair of DSBs, opening the door for further investigations into the enzymes involved in long-range end resection for DSB repair. Double-stranded DNA breaks (DSBs) 2 are one of the most cytotoxic DNA lesions. DSBs can occur naturally inside cells (e.g. meiotic recombination, mating type switch, or V(D)J exchange); they can also be caused by endogenous reactive metabolites and extracellular agents (e.g. UV, IR, and chemical agents such as camptothecin (CPT)) (1). If DSBs are left unrepaired or are repaired inappropriately, it will lead to a variety of mutations such as deletions, insertions, translocation, and genetic fusions, resulting in drastic genomic instability (2, 3).Eukaryotic cells have two major and conserved pathways to repair DSBs: non-homologous end joining (NHEJ) and homologous recombination (HR) (4). In NHEJ, two broken DNA ends are rejoined by the concerted actions of Ku70/80, DNAPKcs, DNA ligase IV, and some other proteins and enzymes (5). NHEJ does not need a homologous DNA sequence for the repair of DSBs, so it can occur in any phase of the cell cycles but dominates at G 0 /G 1 (6). Compared with HR, NHEJ is an errorprone pathway because genetic deletion or insertion of a few base pairs is frequently observed at the break-rejoining site (7,8). HR requires a homologous DNA template that is present in a sister chromatid or an ectopic DNA sequence to fix breaks. Therefore, the HR pathway dominates at the S and G 2 phases and is a relatively error-free repair pathway, although genetic mutations can occur at times (9, 10). A central step in the HR pathway is the 5Ј to 3Ј digestion on one DNA strand from broken DNA ends to generate 3Ј-ended single-stranded DNA (ssDNA) overhangs, a process termed DNA end resection (11,12). The HR-mediated repair of DSBs is reliant on this end resection because only ssDNA is able to invade a homologou...

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“…Extensive resection of DNA ends commits cells to HR, by generating 3 0 -DNA overhangs that are the required substrate for the strand-exchange reaction promoted by the RAD51 recombinase. 2 CtIP (CtBP-interacting protein), also known as RBBP8 (Retinoblastoma-binding protein 8), is an evolutionarily conserved DNA-repair factor with a critical role in HR. 3 It is now well established that a shared function of CtIP and its orthologues in HR-mediated DSB repair is to promote DNA-end resection 4,5 in a cellcycle dependent fashion.…”

Section: Introductionmentioning

confidence: 99%