A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci

Helchowski, Corey M.; Skow, Laura F; Roberts, Katelyn H.; Chute, Colleen L.; Canman, Christine E.

doi:10.4161/cc.26640

Cited by 22 publications

(47 citation statements)

References 47 publications

(55 reference statements)

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“…We also show that RAD18 binds NCP H2AK15ub with high affinity and specificity. This finding unveils a recognition mechanism where a compact domain in RAD18 contacts both ubiquitin and conjugated substrate, and explains how RAD18 accumulates at DSBs (Huang et al, 2009) and interferes with 53BP1 recruitment (Helchowski et al, 2013). Finally, we show that RNF168 reads its lysines 13 and 15 ubiquitylation products, H2AK13ub and H2AK15ub, but unlike RNF169 and RAD18 cannot displace 53BP1 from the modified NCP, shedding light on the ubiquitin signal amplification mechanism that promotes 53BP1 chromatin recruitment.…”

Section: Introductionmentioning

confidence: 74%

“…It was recently shown that ectopic expression of the DNA damage response E3 ubiquitin ligase RAD18 or its ubiquitin-binding zinc finger (UBZ) domain in mammalian cells inhibited the recruitment of 53BP1 and BRCA1 to DSBs (Helchowski et al, 2013). These observations and in vivo evidence that RAD18 requires chromatin ubiquitylation for localization to DNA damage sites (Huang et al, 2009; Panier et al, 2012) prompted us to probe a possible interaction between RAD18 UBZ and the NCP ubiquitylated by RNF168.…”

Section: Resultsmentioning

confidence: 99%

“…53BP1 recruitment is terminated by DNA repair factors RNF169 and RAD18 but the underlying mechanisms are not known (Chen et al, 2012; Helchowski et al, 2013; Panier et al, 2012; Poulsen et al, 2012). Here, we show that RNF169 specifically binds NCP H2AK15ubH4K20me2 with an affinity two orders of magnitude higher than that of 53BP1 for the same substrate.…”

Section: Introductionmentioning

confidence: 99%

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Mechanisms of Ubiquitin-Nucleosome Recognition and Regulation of 53BP1 Chromatin Recruitment by RNF168/169 and RAD18

et al. 2017

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Summary The protein 53BP1 plays a central regulatory role in DNA double-strand break repair. 53BP1 relocates to chromatin by recognizing RNF168-mediated mono-ubiquitylation of histone H2A Lys15 in the nucleosome core particle dimethylated at histone H4 Lys20 (NCP-ubme). 53BP1 relocation is terminated by ubiquitin ligases RNF169 and RAD18 via unknown mechanisms. Using NMR spectroscopy and biochemistry, we show that RNF169 bridges ubiquitin and histone surfaces, stabilizing a pre-existing ubiquitin orientation in NCP-ubme to form a high-affinity complex. This conformational selection mechanism contrasts with the low-affinity binding mode of 53BP1 and ensures 53BP1 displacement by RNF169 from NCP-ubme. We also show that RAD18 binds tightly to NCP-ubme through a ubiquitin-binding domain that contacts ubiquitin and nucleosome surfaces accessed by 53BP1. Our work uncovers diverse ubiquitin recognition mechanisms in the nucleosome, explaining how RNF168, RNF169 and RAD18 regulate 53BP1 chromatin recruitment and how specificity can be achieved in the recognition of a ubiquitin-modified substrate.

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Section: Introductionmentioning

confidence: 74%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Mechanisms of Ubiquitin-Nucleosome Recognition and Regulation of 53BP1 Chromatin Recruitment by RNF168/169 and RAD18

et al. 2017

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“…27,29,30,[32][33][34][35][36][37]57,58 The importance of recognition of methylated histones H3 or H4 by the tudor domain of 53BP1 is well-established. 18,19,22,59 Further, there is universal consensus that RNF8 and RNF168 are required for formation of 53BP1 IRIF.…”

Section: Discussionmentioning

confidence: 99%

“…ubiquitin conjugates, [30][31][32] and overexpression of RAD18 ubiquitin-binding domain blocks RNF168 and 53BP1 accumulation at DNA damage sites. 34 However, 53BP1 lacks any obvious ubiquitin binding motif.…”

Section: Introductionmentioning

confidence: 99%

Ubiquitin-H2AX fusions render 53BP1 recruitment to DNA damage sites independent of RNF8 or RNF168

et al. 2015

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The mammalian E3 ubiquitin ligases RNF8 and RNF168 facilitate recruitment of the DNA damage response protein 53BP1 to sites of DNA double-strand breaks (DSBs). The mechanism involves recruitment of RNF8, followed by recruitment of RNF168, which ubiquitinates histones H2A/H2AX on K15. 53BP1 then binds to nucleosomes at sites of DNA DSBs by recognizing, in addition to methyl marks, histone H2A/H2AX ubiquitinated on K15. We report here that expressing H2AX fusion proteins with N-terminal bulky moieties can rescue 53BP1 recruitment to sites of DNA DSBs in cells lacking RNF8 or RNF168 or in cells treated with proteasome inhibitors, in which histone ubiquitination at sites of DNA DSBs is compromised. The rescue required S139 at the C-terminus of the H2AX fusion protein and was occasionally accompanied by partial rescue of ubiquitination at sites of DNA DSBs. We conclude that recruitment of 53BP1 to sites of DNA DSBs is possible in the absence of RNF8 or RNF168, but still dependent on chromatin ubiquitination.

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RAD18 may function as a predictor of response to preoperative concurrent chemoradiotherapy in patients with locally advanced rectal cancer through caspase‐9‐caspase‐3‐dependent apoptotic pathway

et al. 2019

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Neoadjuvant chemoradiotherapy (nCRT) has been widely applied to improve the local control rate and survival rate in patients with locally advanced rectal cancer (LARC), yet only part of LARC patients would benefit from nCRT. Therefore, it is imperative to predict the therapeutic outcome of nCRT. Here, we showed that RAD18, an E3 ubiquitin‐linked enzyme, played a fundamental role in predicting the response of LARC patients to nCRT. According to clinical data, patients with low RAD18 expression level in their pre‐nCRT biopsies had a superior response to nCRT compared to those with high RAD18 expression. Inhibition of RAD18 expression in rectal cancer cells pronouncedly attenuated the proliferation and promoted apoptosis after exposing to irradiation or/and 5‐fluorouracil (5‐Fu). Downregulated RAD18 levels increased cell apoptosis by activating caspase‐9‐caspase‐3‐mediated apoptotic pathway, thus resulting in the enhancement of cell radiosensitivity and 5‐Fu susceptibility. Furthermore, a xenograft nude mouse model showed that silencing RAD18 significantly slowed tumor growth after irradiation or/and 5‐Fu in vivo. Collectively, these results implied that RAD18 could be a new biomarker to predict LARC patients who might benefit from nCRT and provide new strategies for clinical treatment of LARC.

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A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci

Cited by 22 publications

References 47 publications

Mechanisms of Ubiquitin-Nucleosome Recognition and Regulation of 53BP1 Chromatin Recruitment by RNF168/169 and RAD18

Mechanisms of Ubiquitin-Nucleosome Recognition and Regulation of 53BP1 Chromatin Recruitment by RNF168/169 and RAD18

Ubiquitin-H2AX fusions render 53BP1 recruitment to DNA damage sites independent of RNF8 or RNF168

RAD18 may function as a predictor of response to preoperative concurrent chemoradiotherapy in patients with locally advanced rectal cancer through caspase‐9‐caspase‐3‐dependent apoptotic pathway

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