Ubiquitin-dependent signaling during the DNA damage response (DDR) to double-strand breaks (DSBs) is initiated by two E3 ligases, RNF8 and RNF168, targeting histone H2A and H2AX. RNF8 is the first ligase recruited to the damage site, and RNF168 follows RNF8-dependent ubiquitination. This suggests that RNF8 initiates H2A/H2AX ubiquitination with K63-linked ubiquitin chains and RNF168 extends them. Here, we show that RNF8 is inactive toward nucleosomal H2A, whereas RNF168 catalyzes the monoubiquitination of the histones specifically on K13-15. Structure-based mutagenesis of RNF8 and RNF168 RING domains shows that a charged residue determines whether nucleosomal proteins are recognized. We find that K63 ubiquitin chains are conjugated to RNF168-dependent H2A/H2AX monoubiquitination at K13-15 and not on K118-119. Using a mutant of RNF168 unable to target histones but still catalyzing ubiquitin chains at DSBs, we show that ubiquitin chains per se are insufficient for signaling, but RNF168 target ubiquitination is required for DDR.

Protein ubiquitination is critical for numerous cellular functions, including DNA damage response pathways. Histones are the most abundant monoubiquitin conjugates in mammalian cells; however, the regulation and the function of monoubiquitinated H2A (uH2A) and H2B (uH2B) remain poorly understood. In particular, little is known about mammalian deubiquitinating enzymes (DUBs) that catalyze the removal of ubiquitin from uH2A/uH2B. Here we identify the ubiquitin-specific protease 3 USP3 as a deubiquitinating enzyme for uH2A and uH2B. USP3 dynamically associates with chromatin and deubiquitinates H2A/H2B in vivo. The ZnF-UBP domain of USP3 mediates uH2A-USP3 interaction. Functional ablation of USP3 by RNAi leads to delay of S phase progression and to accumulation of DNA breaks, with ensuing activation of DNA damage checkpoint pathways. In addition, we show that in response to ionizing radiation, (1) uH2A redistributes and colocalizes in gamma-H2AX DNA repair foci and (2) USP3 is required for full deubiquitination of ubiquitin-conjugates/uH2A and gamma-H2AX dephosphorylation. Our studies identify USP3 as a novel regulator of H2A and H2B ubiquitination, highlight its role in preventing replication stress, and suggest its involvement in the response to DNA double-strand breaks. Together, our results implicate USP3 as a novel chromatin modifier in the maintenance of genome integrity.

DNA damage activates signaling pathways that lead to modification of local chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins having ubiquitin ligase activity are recruited to sites of DNA damage, where they ubiquitinate histones and other substrates. This DNA damage-induced histone ubiquitination is thought to play a critical role in mediating the DNA damage response. We now report that the polycomb protein BMI1 is rapidly recruited to sites of DNA damage, where it persists for more than 8 h. The sustained localization of BMI1 to damage sites is dependent on intact ATM and ATR and requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads to impaired repair of DNA double-strand breaks by homologous recombination and the accumulation of cells in G 2 /M. These data support a crucial role for BMI1 in the cellular response to DNA damage.The induction of a DNA break leads to activation of multiple signaling pathways that lead to local modification of chromatin structure and recruitment of DNA repair complexes (18,22,55). Histone H2AX is rapidly phosphorylated near sites of DNA breaks by ATM, ATR, and DNA-PK (39, 54) and can spread to encompass a region of chromatin covering several megabases (40, 41).H2AX phosphorylation facilitates the recruitment of other proteins, including MDC1 (52) and the E3 ubiquitin ligases RNF8 and RNF168, which in turn participate locally in the K63-linked polyubiquitination of histones H2A and H2AX (23,32,50,51). Polyubiquitinated K63-linked histones provide a recognition element that recruits RAP80 through its ubiquitin interaction motifs (28,49,56). RAP80 can then promote the recruitment of other DNA repair factors such as BRCA1 and Abraxas, which are essential for efficient repair. RNF8 and RNF168 function are also required for proper localization of 53BP1, although the exact mechanism is unclear (12,23,32,51). 53BP1 recruitment to regions of DNA damage is dependent upon its Tudor domains, which have been found to specifically interact with methylated histone residues (6, 24, 42). A model has been proposed in which RNF8-and RNF168-mediated ubiquitination of histones confers local changes in chromatin structure, leading to exposure of methylated lysine residues in core histones, allowing the subsequent recruitment of 53BP1 (50). Enzymes involved in deubiquitination, such as BRCC36, USP3, and USP28, are also critical for efficient DNA repair, demonstrating that a dynamic regulation of ubiquitin conjugation and hydrolysis is necessary for optimal DNA repair (37,46,47,61).Polycomb group proteins BMI1 and RING1B/RNF2 form an active heterodimer E3 ligase that catalyzes the monoubiquitination of histone H2A at Lysine 119. (7,8,44,53,57). This activity is important for BMI1-mediated transcriptional silencing during organism development and cellular differentiation (27,48,58). Ubiquitination of H2A at lysine 119 is also induced locally at sites of DNA damage, both at s...

The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/ SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed.

Chromatin changes within the context of DNA repair remain largely obscure. Here we show that DNA damage induces monoubiquitylation of histone H2A in the vicinity of DNA lesions. Ultraviolet (UV)-induced monoubiquitylation of H2A is dependent on functional nucleotide excision repair and occurs after incision of the damaged strand. The ubiquitin ligase Ring2 is required for the DNA damage-induced H2A ubiquitylation. UV-induced ubiquitylation of H2A is dependent on the DNA damage signaling kinase ATR (ATM-and Rad3-related) but not the related kinase ATM (ataxia telangiectasia-mutated). Although the response coincides with phosphorylation of variant histone H2AX, H2AX was not required for H2A ubiquitylation. Together our data show that monoubiquitylation of H2A forms part of the cellular response to UV damage and suggest a role of this modification in DNA repair-induced chromatin remodeling.[Keywords: DNA repair; ubiquitin-proteasome system; histone; chromatin; ATR; H2AX; DNA damage response] Supplemental material is available at http://www.genesdev.org.

Transcription‐coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair‐ and transcription‐competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10–15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell‐free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency.

Error-free repair by homologous recombination of DNA double-strand breaks induced by ionizing radiation (IR) requires the Rad52 group proteins, including Rad51 and Rad54, in the yeast Saccharomyces cerevisiae [1]. The formation of a 'joint' molecule between the damaged DNA and the homologous repair template is a key step in recombination mediated by Rad51 and stimulated by Rad54 [2] [3] [4] [5]. Mammalian homologs of Rad51 and Rad54 have been identified [2] [3] [6]. Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci that colocalized with mRad51. Interaction between mRad51 and mRad54 was induced by genotoxic stress, but only when lesions that required mRad54 for their repair were formed. Interestingly, mRad54 was essential for the formation of IR-induced mRad51 foci. Rad54 belongs to the SWI2/SNF2 protein family, members of which modulate protein-DNA interactions in an ATP-driven manner [7]. Results of a topological assay suggested that purified human Rad54 (hRad54) protein can unwind double-stranded (ds) DNA at the expense of ATP hydrolysis. Unwinding of the homologous repair template could promote the formation or stabilization of hRad51-mediated joint molecules. Rad54 appears to be required downstream of other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that assist Rad51 in interacting with the broken DNA [2] [3] [4].

Np95 is an important determinant in cell cycle progression. Its expression is tightly regulated and becomes detectable shortly before the entry of cells into S phase. Accordingly, Np95 is absolutely required for the G 1 /S transition. Its continued expression throughout the S/G 2 /M phases further suggests additional roles. Indeed, Np95 has been implicated in DNA damage response. Here, we show that Np95 is tightly bound to chromatin in vivo and that it binds to histones in vivo and in vitro. The binding to histones is direct and shows a remarkable preference for histone H3 and its N-terminal tail. A novel protein domain, the SRA-YDG domain, contained in Np95 is indispensable both for the interaction with histones and for chromatin binding in vivo. Np95 contains a RING finger. We show that this domain confers E3 ubiquitin ligase activity on Np95, which is specific for core histones, in vitro. Finally, Np95 shows specific E3 activity for histone H3 when the endogenous core octamer, coimmunoprecipitating with Np95, is used as a substrate. Histone ubiquitination is an important determinant in the regulation of chromatin structure and gene transcription. Thus, the demonstration that Np95 is a chromatin-associated ubiquitin ligase suggests possible molecular mechanisms for its action as a cell cycle regulator.Ubiquitination is a frequent posttranslational modification with a vast impact on cell physiology. Ubiquitin (Ub) is a conserved 76-amino-acid polypeptide that is covalently attached to target proteins via an isopeptide bond between its carboxyl-terminal glycine and the ε-amino group of a lysine in substrate proteins (23). A complex enzymatic cascade leads to ubiquitination. Ub is first activated through the formation of a thiol ester bond with the Ub-activating enzyme (E1) and then transferred to a Ub-conjugating enzyme (E2). Finally, Ub is transferred to the substrate through the action of a Ub ligase (E3). Two major families of E3s exist, the HECT type and the RING type. In HECT-E3-mediated catalysis, Ub is transferred from E2 to HECT-E3 and then by E3 to the substrate. In RING-E3-mediated catalysis, E3 mediates the direct transfer of Ub from E2 to the substrate. The E3 ligases, therefore, are the substrate recognition components of the system and confer specificity on the process (23, 47).The best-characterized type of ubiquitination is polyubiquitination, in which the substrate-bound Ub serves as an acceptor for further cycles of ubiquitination (23). By and large, polyubiquitin functions as a general device for targeting of the polyubiquitinated substrate to the proteasome, with ensuing proteolytic degradation (25,48). An emerging body of evidence indicates, however, that when Ub is appended as a single moiety to a target protein (monoubiquitination), the posttranslational modification has a completely different biological impact and serves primarily to modulate protein function and/or interaction(s) (10,24,57).Histones are among the major monoubiquitinated proteins in the cell, and histones H2A, H2B, and H3...