ATM-dependent Phosphorylation of the Fanconi Anemia Protein PALB2 Promotes the DNA Damage Response

Guo, Yingying; Feng, Wei; Sy, Shirley M.-H.; Huen, Michael S.Y.

doi:10.1074/jbc.m115.672626

Cited by 19 publications

(21 citation statements)

References 41 publications

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“…Consistent with previous reports (23,24), siRNA-mediated depletion of BRCC36 led to increased rates of HR repair (Fig. 1B), whereas inactivation of the HR repair factor PALB2 led to a marked reduction in DSB repair events (26). To directly examine how the BRCC36 DUB contributes to DSB repair, we generated cells that stably express siRNA-resistant versions of BRCC36 WT or its DUB enzymatic mutant BRCC36 H122Q/H124Q (QSQ).…”

Section: Resultssupporting

confidence: 83%

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: Resultssupporting

confidence: 83%

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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“…We identified a total of 12 phosphorylation sites on PALB2 (Fig. 3A), a subset of which was shown previously (Ahlskog et al, 2016; Guo et al, 2015; Matsuoka et al, 2007). The N-terminal coiled-coil domain of PALB2 interacts with BRCA1 directly (Fig.…”

Section: Resultssupporting

confidence: 69%

“…ATR may have additional roles in PALB2 regulation. A recent study suggested that the phosphorylation of PALB2 at S157 and S376, two putative ATM/ATR sites, is dispensable for HR but required for the recovery from the DNA damage response (Guo et al, 2015). Another study proposed that the phosphorylation of PALB2 by ATM/ATR at S59, S157, and S376 promotes HR by enhancing the interaction between PALB2 and RAD51 (Ahlskog et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

Coupling of Homologous Recombination and the Checkpoint by ATR

et al. 2017

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ATR is a key regulator of cell cycle checkpoints and homologous recombination (HR). Paradoxically, ATR inhibits CDKs during checkpoint responses, but CDK activity is required for efficient HR. Here, we show that ATR promotes HR after CDK-driven DNA end resection. ATR stimulates the BRCA1-PALB2 interaction after DNA damage, and promotes PALB2 localization to DNA damage sites. ATR enhances BRCA1-PALB2 binding at least in part by inhibiting CDKs. The optimal interaction of BRCA1 and PALB2 requires phosphorylation of PALB2 at S59, an ATR site, and hypo-phosphorylation of S64, a CDK site. The PALB2-S59A/S64E mutant is defective for localization to DNA damage sites and HR, whereas the PALB2-S59E/S64A mutant partially bypasses ATR for its localization. Thus, HR is a biphasic process requiring both high-CDK and low-CDK periods. As exemplified by the regulation of PALB2 by ATR, ATR promotes HR by orchestrating a “CDK-to-ATR switch” post resection, directly coupling the checkpoint with HR.

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“…U2OS cells were infected with lentiviral particles carrying RNF169-SFB expression constructs and were subsequently selected by 2 mg/ mL G418. Expression of RNF169-SFB was induced by supplementing cell culture media with doxycycline at 1 μg/mL for 12 h. Generation of cell lines with constitutive expression of RNF169 (wild type and mutants) and USP7 (wild type and mutants) was performed as previously described (50).…”

Section: Methodsmentioning

confidence: 99%

“…Procedures for indirect immunofluorescence experiments were as previously described (50). Briefly, cells grown as monolayers on coverslips were exposed to ionizing radiation (10 Gy).…”

Section: Methodsmentioning

confidence: 99%

Dual-utility NLS drives RNF169-dependent DNA damage responses

Jiang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Loading of p53-binding protein 1 (53BP1) and receptor-associated protein 80 (RAP80) at DNA double-strand breaks (DSBs) drives cell cycle checkpoint activation but is counterproductive to high-fidelity DNA repair. ring finger protein 169 (RNF169) maintains the balance by limiting the deposition of DNA damage mediator proteins at the damaged chromatin. We report here that this attribute is accomplished, in part, by a predicted nuclear localization signal (NLS) that not only shuttles RNF169 into the nucleus but also promotes its stability by mediating a direct interaction with the ubiquitin-specific protease USP7. Guided by the crystal structure of USP7 in complex with the RNF169 NLS, we uncoupled USP7 binding from its nuclear import function and showed that perturbing the USP7-RNF169 complex destabilized RNF169, compromised high-fidelity DSB repair, and hypersensitized cells to poly (ADP-ribose) polymerase inhibition. Finally, expression of USP7 and RNF169 positively correlated in breast cancer specimens. Collectively, our findings uncover an NLS-mediated bipartite mechanism that supports the nuclear function of a DSB response protein.ells respond to DNA double-strand breaks (DSBs) by mounting a series of signal transduction events that culminate in cell arrest and DNA repair (1). Signal transduction entails the coordinated assembly of a cohort of DNA damage mediator proteins, including p53-binding protein 1 (53BP1) and receptor-associated protein 80 (RAP80), at the damaged chromatin, which, in turn, enforces checkpoint control and cell tolerance to DNA damage (2, 3). Paradoxically, although DSB loading of 53BP1 and RAP80 underlies robust activation of DNA damage responses (DDRs), they operate at the expense of high-fidelity DNA repair, because their productive accumulation at DSB-flanking chromatin blocks DNA end resection and suppresses RAD51-dependent homologous recombination (HR) repair (4-11). Exactly how cells achieve a balance between robust DSB signal transduction and HR repair remains to be defined, but several lines of evidence converge on the idea that limiting the extent of DSB ubiquitylation may selectively promote HR repair (12-15), highlighting the need for cell-intrinsic mechanisms to restrain chromatin responses arising from DSBs (16).Intriguingly, one such cell-intrinsic strategy involves ring finger protein 169 (RNF169), a paralog of the RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties) syndrome protein RNF168 (17,18). In stark contrast to RNF168, which plays positive roles in amplifying ubiquitin-dependent DSB signals (18,19), RNF169 is endowed with antagonistic properties in the DSB signal transduction pathway. Indeed, RNF169 competes for RNF168-catalyzed ubiquitin adducts and displaces 53BP1 and RAP80 from DSBs (14,20,21). Despite its putative role as an integral component of the DSB signal transduction cascade, how RNF169 is regulated mechanistically and how it contributes to the multipartite self-restraining mechanisms in DNA damage surveillance and...

show abstract

ATM-dependent Phosphorylation of the Fanconi Anemia Protein PALB2 Promotes the DNA Damage Response

Cited by 19 publications

References 41 publications

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

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

Coupling of Homologous Recombination and the Checkpoint by ATR

Dual-utility NLS drives RNF169-dependent DNA damage responses

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