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The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair.

ABSTRACT: The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdc1. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 as a novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage.

SUBMITTER: Xu Y

PROVIDER: S-EPMC2953432 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair.

Xu Ye Y Sun Yingli Y Jiang Xiaofeng X Ayrapetov Marina K MK Moskwa Patryk P Yang Shenghong S Weinstock David M DM Price Brendan D BD

The Journal of cell biology 20100927 1

The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity o ...[more]

PMID: 20876283

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Project description:BackgroundMaintenance of genome integrity during DNA replication is crucial to the perpetuation of all organisms. In eukaryotes, the bypass of DNA lesions by the replication machinery prevents prolonged stalling of the replication fork, which could otherwise lead to greater damages such as gross chromosomal rearrangements. Bypassing DNA lesions and subsequent repair are accomplished by the activation of DNA damage tolerance pathways such as the template switching (TS) pathway. In yeast, the RAD5 (Radiation-sensitive 5) protein plays a crucial role in initiating the TS pathway by catalyzing the polyubiquitination of PCNA (Proliferation Cell Nuclear Antigen). Likewise, one of the mammalian RAD5-homologs, SHPRH (SNF2, histone linker, PHD, RING, helicase) mediates PCNA polyubiquitination. To date, the study of SHPRH enzymatic functions has been limited to this modification. It is therefore unclear how SHPRH carries out its function in DNA repair. Moreover, how this protein regulates gene transcription at the enzymatic level is also unknown.ResultsGiven that SHPRH harbors domains found in chromatin remodeling proteins, we investigated its biochemical properties in the presence of nucleosomal substrates. We find that SHPRH binds equally well to double-stranded (ds) DNA and to nucleosome core particles, however, like ISWI and CHD-family remodelers, SHPRH shows a strong preference for nucleosomes presenting extranucleosomal DNA. Moreover, nucleosomes but not dsDNA strongly stimulate the ATPase activity of SHPRH. Intriguingly, unlike typically observed with SNF2-family enzymes, ATPase activity does not translate into conventional nucleosome remodeling, under standard assay conditions. To test whether SHPRH can act as a ubiquitin E3 ligase for nucleosomes, we performed a screen using 26 E2-conjugating enzymes. We uncover that SHPRH is a potent nucleosome E3-ubiquitin-ligase that can function with at least 7 different E2s. Mass spectrometry analyses of products generated in the presence of the UBE2D1-conjugating enzyme reveal that SHPRH can catalyze the formation of polyubiquitin linkages that are either branched or associated with the recruitment of DNA repair factors, as well as linkages involved in proteasomal degradation.ConclusionsWe propose that, in addition to polyubiquitinating PCNA, SHPRH promotes DNA repair or transcriptional regulation in part through chromatin ubiquitination. Our study sets a biochemical framework for studying other RAD5- and RAD16-related protein functions through the ubiquitination of nucleosomes.

Project description:Recent reports have made important revelations, uncovering direct regulation of DNA damage response (DDR)-associated proteins and chromatin ubiquitination (Ubn) by macroautophagy/autophagy. Here, we report a previously unexplored connection between autophagy and DDR, via a deubiquitnase (DUB), USP14. Loss of autophagy in prostate cancer cells led to unrepaired DNA double-strand breaks (DSBs) as indicated by persistent ionizing radiation (IR)-induced foci (IRIF) formation for γH2AFX, and decreased protein levels and IRIF formation for RNF168, an E3-ubiquitin ligase essential for chromatin Ubn and recruitment of critical DDR effector proteins in response to DSBs, including TP53BP1. Consistently, RNF168-associated Ubn signaling and TP53BP1 IRIF formation were reduced in autophagy-deficient cells. An activity assay identified several DUBs, including USP14, which showed higher activity in autophagy-deficient cells. Importantly, inhibiting USP14 could overcome DDR defects in autophagy-deficient cells. USP14 IRIF formation and protein stability were increased in autophagy-deficient cells. Co-immunoprecipitation and colocalization of USP14 with MAP1LC3B and the UBA-domain of SQSTM1 identified USP14 as a substrate of autophagy and SQSTM1. Additionally, USP14 directly interacted with RNF168, which depended on the MIU1 domain of RNF168. These findings identify USP14 as a novel substrate of autophagy and regulation of RNF168-dependent Ubn and TP53BP1 recruitment by USP14 as a critical link between DDR and autophagy. Given the role of Ubn signaling in non-homologous end joining (NHEJ), the major pathway for repair of IR-induced DNA damage, these findings provide unique insights into the link between autophagy, DDR-associated Ubn signaling and NHEJ DNA repair.AbbreviationsATG7: autophagy related 7; CQ: chloroquine; DDR: DNA damage response; DUB: deubiquitinase; HR: homologous recombination; IR: ionizing radiation; IRIF: ionizing radiation-induced foci; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MIU1: motif interacting with ubiquitin; NHEJ: non homologous end-joining; PCa: prostate cancer; TP53BP1/53BP1: tumor protein p53 binding protein 1; RNF168: ring finger protein 168; SQSTM1/p62 sequestosome 1; γH2AFX/γH2AX: H2A histone family member X: phosphorylated, UBA: ubiquitin-associated; Ub: ubiquitin; Ubn: ubiquitination; USP14: ubiquitin specific peptidase 14.

Dataset Information

The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair.

Publications

The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair.

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