RNF168 forms a functional complex with RAD6 during the DNA damage response

Liu, Chao; Wang, Degui; Wu, Jiaxue; Keller, Jonathan M.; Ma, Teng; Yu, Xiaochun

doi:10.1242/jcs.122945

Cited by 35 publications

(29 citation statements)

References 62 publications

(96 reference statements)

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“…Our results suggest that this is likely because UbcH7-depleted cells express elevated levels of 53BP1, allowing the formation of more and larger 53BP1 foci at DSB sites. The E2 enzyme RAD6 indirectly regulates 53BP1 foci through modulating RNF168 (41). Here, our results revealed that regulating 53BP1 levels has as strong impact as regulating the foci formation of 53BP1 on the DSB repair choice.…”

Section: Discussionmentioning

confidence: 56%

UbcH7 regulates 53BP1 stability and DSB repair

Han¹,

Zhang²,

Chung³

et al. 2014

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

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DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the errorprone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.DNA damage response | UbcH7 | 53BP1 | protein degradation | DSB repair P rompt response to double-strand breaks (DSBs) caused by, for example, ionization radiation (IR), requires sequential and coordinated assembly of DNA damage response (DDR) proteins at damage sites (1). Recent research findings reveal key roles of the tumor suppressor p53-binding protein 1 (53BP1) and BRCA1 in the decision making of DSB repair. 53BP1, together with Rif1, suppress BRCA1-dependent homologous recombination (HR), thereby promoting nonhomologous end-joining (NHEJ) in G1 phase (2-6). Conversely, BRCA1 antagonizes 53BP1/Rif1, favoring HR in S and G2 phases (7,8). In the absence of BRCA1 or with enhanced retention of 53BP1 at DSB sites, cells primarily use the error-prone NHEJ to repair DSBs throughout the cell cycle, which leads to gene rearrangement, cell death, and increased sensitivity to anticancer therapies (9-11). Consistently, BRCA1-null mice are early embryonic lethal (12, 13) and codepletion of TP53BP1 rescued the lethality phenotype of BRCA1-null mice (12)(13)(14).Low expression level of 53BP1 was found to be associated with poor clinical outcome in triple negative breast cancer patients with BRCA1 mutation (12, 15), as well as resistance to genotoxins and poly(ADP-ribose) polymerase inhibitors (12,16,17). This finding is probably because loss of 53BP1 restored HR and promoted cell survival (12-14). Reduced expression of 53BP1 was also observed in tumors from the brain (18), lymph node (19), and pancreas (20). These data indicate that loss of 53BP1 might be a common mechanism for advanced tumors to evade from radiotherapy or chemotherapy. However, molecular mechanisms controlling the protein level of 53BP1 remain less well understood.Here we show that UbcH7, an E2 enzyme involved in the ubiquitin (Ub) pathway, controls the protein stability of 53BP1, thereby determining the DSB repair choice. Loss of UbcH7 sta...

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Section: Discussionmentioning

confidence: 56%

UbcH7 regulates 53BP1 stability and DSB repair

Han¹,

Zhang²,

Chung³

et al. 2014

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

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“…Recently, RNF168 has also been shown to interact with the A and B isoforms of the E2 ubiquitin-conjugating enzyme RAD6 (37). These are orthologs of yeast Rad6, which helps mediate postreplication DNA repair, proteolysis via the N-end rule pathway, and other processes (38).…”

Section: Upstream Action Of Rnf8 and Rnf168 Ubiquitin Ligasesmentioning

confidence: 99%

“…Both RAD6 isoforms are required for 53BP1 and BRCA1 recruitment to DSBs (37). Liu et al (37) implicated the RAD6A/B-RNF168 complexes in ubiquitination of the linker histone H1.2. However, the biological relevance of H1.2 ubiquitination remains to be established.…”

Section: Upstream Action Of Rnf8 and Rnf168 Ubiquitin Ligasesmentioning

confidence: 99%

53BP1, BRCA1, and the Choice between Recombination and End Joining at DNA Double-Strand Breaks

Daley

Sung

2014

Molecular and Cellular Biology

246

240

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When DNA double-strand breaks occur, the cell cycle stage has a major influence on the choice of the repair pathway employed. Specifically, nonhomologous end joining is the predominant mechanism used in the G 1 phase of the cell cycle, while homologous recombination becomes fully activated in S phase. Studies over the past 2 decades have revealed that the aberrant joining of replication-associated breaks leads to catastrophic genome rearrangements, revealing an important role of DNA break repair pathway choice in the preservation of genome integrity. 53BP1, first identified as a DNA damage checkpoint protein, and BRCA1, a well-known breast cancer tumor suppressor, are at the center of this choice. Research on how these proteins function at the DNA break site has advanced rapidly in the recent past. Here, we review what is known regarding how the repair pathway choice is made, including the mechanisms that govern the recruitment of each critical factor, and how the cell transitions from end joining in G 1 to homologous recombination in S/G 2 .

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“…In vitro ubiquitylation assays were performed as described previously (Mattiroli et al 2012;Fradet-Turcotte et al 2013;Liu et al 2013). Briefly, 2.5 µg of mononucleosomes was incubated with 30 nM yeast E1, 1.5 µM UbcH5a, 4 µM RNF168 (1-113), 10 µM Flag-ubiquitin, and 3 mM ATP in ubiquitylation buffer (50 mM Tris-HCl at pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 1 mM ZnCl 2 , 1 mM DTT) for 2 h at 30°C.…”

Section: In Vitro Ubiquitylation and Deubiquitylation Assaysmentioning

confidence: 99%

USP51 deubiquitylates H2AK13,15ub and regulates DNA damage response

et al. 2016

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Dynamic regulation of RNF168-mediated ubiquitylation of histone H2A Lys13,15 (H2AK13,15ub) at DNA doublestrand breaks (DSBs) is crucial for preventing aberrant DNA repair and maintaining genome stability. However, it remains unclear which deubiquitylating enzyme (DUB) removes H2AK13,15ub. Here we show that USP51, a previously uncharacterized DUB, deubiquitylates H2AK13,15ub and regulates DNA damage response. USP51 depletion results in increased spontaneous DNA damage foci and elevated levels of H2AK15ub and impairs DNA damage response. USP51 overexpression suppresses the formation of ionizing radiation-induced 53BP1 and BRCA1 but not RNF168 foci, suggesting that USP51 functions downstream from RNF168 in DNA damage response. In vitro, USP51 binds to H2A-H2B directly and deubiquitylates H2AK13,15ub. In cells, USP51 is recruited to chromatin after DNA damage and regulates the dynamic assembly/disassembly of 53BP1 and BRCA1 foci. These results show that USP51 is the DUB for H2AK13,15ub and regulates DNA damage response.

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RNF168 forms a functional complex with RAD6 during the DNA damage response

Cited by 35 publications

References 62 publications

UbcH7 regulates 53BP1 stability and DSB repair

UbcH7 regulates 53BP1 stability and DSB repair

53BP1, BRCA1, and the Choice between Recombination and End Joining at DNA Double-Strand Breaks

USP51 deubiquitylates H2AK13,15ub and regulates DNA damage response

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