A Critical Role for the Ubiquitin-Conjugating Enzyme Ubc13 in Initiating Homologous Recombination

Zhao, Guang Yu; Sonoda, Eiichiro; Barber, Louise J.; Oka, Hayato; Murakawa, Yuji; Yamada, Kazutoyo; Ikura, Tsuyoshi; Wang, Xin; Kobayashi, Masahiko; Yamamoto, Kenichi; Boulton, Simon J.; Takeda, Shunichi

doi:10.1016/j.molcel.2007.01.029

Cited by 202 publications

(222 citation statements)

References 49 publications

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“…Our results showed that the knockdown of SHPRH or HLTF also increased the frequency of mutation in Ramos cells (Fig. 4A), suggesting that a reduction of (23,24,34). Consistent with these results, we also observed that the depletion of MMS2 in human U2OS/DRGFP cells reduced HR efficiency (Fig.…”

Section: Discussionsupporting

confidence: 88%

“…Yeast Ubc13 and Mms2 are exclusively involved in the Rad5 pathway, but not in HR. In contrast, in higher eukaryotes, UBC13 and MMS2 seem to function in HR in addition to their roles in PRR (23,24). To determine whether SHPRH and HLTF are involved in HR, we used a recombination reporter assay (DR-GFP reporter assay), which can measure the frequency of HR between tandem GFP repeats with inactivating mutations (25).…”

Section: Shprh and Hltf Are Not Directly Involved In Homologous Recommentioning

confidence: 99%

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Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks

Motegi

Liaw

Lee

et al. 2008

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

242

269

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C hronic stalling of DNA replication forks by DNA damage such as UV irradiation, ionizing irradiation, chemicals, and reactive cellular metabolites impedes the progression of the cell cycle and eventually causes cell death. To circumvent such situations, cells have evolved the postreplication repair (PRR) pathway that bypasses DNA lesions to resolve stalled forks without removing the actual damage (1). In budding yeast Saccharomyces cerevisiae, PRR is carried out by 2 distinct pathways: translesion synthesis (TLS) and template switching (TS) (Fig. 1A). TLS uses multiple low-fidelity TLS polymerases to incorporate nucleotides across DNA lesions (2, 3). Switching from replicative polymerases ␦ or to TLS polymerases is promoted through the interaction between monoubiquitinated PCNA at lysine 164 (K164) and a ubiquitin-binding motif in TLS polymerases-a mechanism conserved from the budding yeast to human. The monoubiquitination of PCNA at K164 requires the RING-type ubiquitin ligase Rad18 (E3) and the ubiquitinconjugating enzyme Rad6 (E2).The TS pathway bypasses DNA damage by switching a stalled replicating end to the nascent daughter strand of the sister chromatid (1, 4). This pathway involves a lysine 63 (K63)-linked polyubiquitin chain that is further added onto the monoubiquitinated PCNA by Rad5 (E3) along with the Ubc13-Mms2 (E2 and E2 variant, respectively) heterodimer complex (Fig. 1 A). Distinct from the K48-linked polyubiquitination leading to protein degradation, the K63-linked polyubiquitination of PCNA is thought to promote TS in a proteasome-independent manner (5).The importance of the TLS pathway in the suppression of mammalian tumorigenesis emerged with the identification of a mutation in TLS polymerase in patients with the variant form of xeroderma pigmentosum and from studies with mouse models (6, 7). Despite the presence of UBC13 and MMS2 homologues in humans, the importance of the TS pathway is less clear in mammals because K63-linked polyubiquitination of PCNA, a hallmark event for the TS pathway, had not been observed until recently (8-10). We recently identified human SHPRH, which possesses SWI2/SNF2 and RING domains with similar architecture to the yeast Rad5 as a functional homologue of yeast Rad5 (9). Specifically, we demonstrated the in vivo activity of SHPRH in promoting a K63-linked polyubiquitination of PCNA as well as physical interactions of SHPRH with PCNA, RAD18, and UBC13. Depletion of SHPRH increases genomic instability after genotoxic stress. Consistent with our work, another study also demonstrated that SHPRH could polyubiquitinate PCNA in vitro (11).In the present study, we demonstrated that ectopic expression of HLTF/SMARCA3/RUSH/HIP116/Zbu1 (hereafter, HLTF) enhanced PCNA polyubiquitination in vivo. Depletion of SHPRH or HLTF significantly reduced polyubiquitination of chromatin-bound PCNA upon treatment of cells with DNA-damaging agents that cause stalled DNA replication forks. Furthermore, Hltf-deficient mouse embryonic fibroblasts (MEFs) showed elevated chromosome breaks an...

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Section: Discussionsupporting

confidence: 88%

Section: Shprh and Hltf Are Not Directly Involved In Homologous Recommentioning

confidence: 99%

Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks

Motegi

Liaw

Lee

et al. 2008

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

242

269

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“…Following the initial DSB accumulation events, RNF8-Ubc13 and Rap80-BRCC36 are predicted to be present at the same DSB regions, consistent with the hypothesis that both enzymatic complexes simultaneously regulate DSB-associated K63-Ub levels. ␥H2AX has recently been shown to be an RNF8-Ubc13 substrate; thus, ubiquitinated ␥H2AX (␥H2AX-Ub) is a putative recognition element for the Rap80-BRCC36 complex (6,8,17). To determine if Rap80-BRCC36 targets ubiquitinated forms of ␥H2AX during DSB repair responses, cells were treated with control versus BRCC36 siRNA and histones purified by acid elution 1 h after 10 Gy IR.…”

Section: Resultsmentioning

confidence: 99%

“…PI3K-like kinase phosphorylation of MDC1 (13,16) creates a binding site for the E3 ubiquitin ligase RNF8 (Ring Finger 8). In conjunction with the E2 enzyme Ubc13 (ubiquitin-conjugating 13), RNF8 ubiquitinates histones H2A and H2AX in a K63-linked manner to create a docking site for DNA repair proteins to accumulate at DSBs (6)(7)(8)17). These DSB chromatin-associated K63-linked ubiquitin species are recognized by the tandem ubiquitin interacting motif (UIM) containing protein Rap80/UIMC1 (Receptor Associated Protein 80/Ubiquitin Interaction Motif Containing 1) as part of a biochemical complex that includes BRCA1 (9)(10)(11)18).…”

mentioning

confidence: 99%

The Rap80-BRCC36 de-ubiquitinating enzyme complex antagonizes RNF8-Ubc13-dependent ubiquitination events at DNA double strand breaks

Shao¹,

Lilli²,

Patterson-Fortin

et al. 2009

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

200

207

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DNA double strand breaks (DSBs) initiate reversible cellular checkpoint and repair activities. Whereas many of the activating events at DSBs have recently been elucidated, the mechanisms used to terminate responses at these sites are largely undefined. Here we report a pathway required to reverse RNF8-Ubc13 dependent ubiquitination events on chromatin flanking DSBs. Inhibition of the Rap80-BRCC36 de-ubiquitinating enzyme complex partially restored DSB-associated ubiquitin levels following RNF8 knockdown or proteasome inhibition. Similarly, BRCC36 knockdown or expression of a BRCC36 de-ubiquitinating enzyme-inactive mutant rescued both 53BP1 recruitment to DSBs and ionizing radiation-induced ␥H2AX ubiquitination following RNF8 depletion, and mitigated ionizing radiation sensitivity resulting from RNF8 deficiency. Thus, concomitant and opposing RNF8-Ubc13 ubiquitin ligase and Rap80-BRCC36 ubiquitin hydrolysis activities are responsible for determining steady-state ubiquitin levels at DNA DSBs. These findings reveal a Rap80-BRCC36 dependent pathway that is required for appropriate DSB recruitment and repair responses.BRCA1 ͉ DNA damage ͉ ubiquitin M ultiple, parallel signaling pathways converge upon DNA double-strand breaks (DSBs) to initiate temporal and spatial coordination of checkpoint and repair responses. These DNA damage response activities are mediated in part by accumulation of DNA repair proteins at both chromatin and non-nucleosomal DNA regions flanking DSBs (1-3). Repair factor targeting occurs within minutes of DSB induction, providing strong evidence that molecular recognition events rapidly develop at sites of DNA damage (1,4,5). Following the resolution of DNA damage, repair proteins dissociate from DSBs, thus alleviating cell cycle checkpoint responses and allowing resumption of cell proliferation. It is unclear, however, if DSB termination and activation pathways occur simultaneously in an equilibrium process during the earliest stages of repair, or if DNA damage response resolution occurs in a step-wise manner subsequent to the completion of DNA repair.Some of the recruitment events at DSBs have recently been elucidated for the breast and ovarian cancer suppressor protein BRCA1 (Breast Cancer 1, early onset) and the checkpoint and repair protein 53BP1 (p53 Binding Protein 1) (6-12). Histone H2AX phosphorylation by the PI3K-like kinase members ATM (Ataxia Telangiectasia Mutated) and DNA-PKcs (DNADependent Protein Kinase catalytic subunit) occurs at chromatin flanking DSBs to initiate a direct interaction between phosphorylated serine 139 of H2AX (␥H2AX) and the MDC1 (Mediator of DNA Damage Checkpoint Protein 1) protein (13-15). PI3K-like kinase phosphorylation of MDC1 (13, 16) creates a binding site for the E3 ubiquitin ligase RNF8 (Ring Finger 8). In conjunction with the E2 enzyme Ubc13 (ubiquitin-conjugating 13), RNF8 ubiquitinates histones H2A and H2AX in a K63-linked manner to create a docking site for DNA repair proteins to accumulate at DSBs (6-8, 17). These DSB chromatin-associated K63-link...

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“…In vitro reconstitution of this complex with recombinant proteins will be necessary to definitively address these possibilities. PIKK activity has been linked to histone ubiquitylation, the putative DSB-associated ligand for the BRCA1-RAP80 complex (Huen et al, 2007;Kolas et al, 2007;Mailand et al, 2007;Zhao et al, 2007). As mentioned earlier, -H2AX serves as a signal to initiate recruitment of additional factors to DNA repair foci.…”

Section: Connections Between Pikk Activity and Ubiquitylationmentioning

confidence: 98%

The ubiquitin landscape at DNA double-strand breaks

Messick¹,

Greenberg²

2009

J Exp Med

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A Critical Role for the Ubiquitin-Conjugating Enzyme Ubc13 in Initiating Homologous Recombination

Cited by 202 publications

References 49 publications

Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks

Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks

The Rap80-BRCC36 de-ubiquitinating enzyme complex antagonizes RNF8-Ubc13-dependent ubiquitination events at DNA double strand breaks

The ubiquitin landscape at DNA double-strand breaks

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