Differential regulation of RNF8-mediated Lys48- and Lys63-based poly-ubiquitylation

Pairing of a given E3 ubiquitin ligase with different E2s allows synthesis of ubiquitin conjugates of different topologies. While this phenomenon contributes to functional diversity, it remains largely unknown how a single E3 ubiquitin ligase recognizes multiple E2s, and whether identical structural requirements determine their respective interactions. The E3 ubiquitin ligase RNF8 that plays a critically important role in transducing DNA damage signals, interacts with E2s UBCH8 and UBC13, and catalyzes both K48- and K63-linked ubiquitin chains. Interestingly, we report here that a single-point mutation (I405A) on the RNF8 polypeptide uncouples its ability in catalyzing K48- and K63-linked ubiquitin chain formation. Accordingly, while RNF8 interacted with E2s UBCH8 and UBC13, its I405A mutation selectively disrupted its functional interaction with UBCH8, and impaired K48-based poly-ubiquitylation reactions. In contrast, RNF8 I405A preserved its interaction with UBC13, synthesized K63-linked ubiquitin chains, and assembled BRCA1 and 53BP1 at sites of DNA breaks. Together, our data suggest that RNF8 regulates K48- and K63-linked poly-ubiquitylation via differential RING-dependent interactions with its E2s UBCH8 and UBC13, respectively.published_or_final_versio

BRCA1 and its toolbox for the maintenance of genome integrity

The breast and ovarian cancer type 1 susceptibility protein (BRCA1) has pivotal roles in the maintenance of genome stability. Studies support that BRCA1 exerts its tumour suppression function primarily through its involvement in cell cycle checkpoint control and DNA damage repair. In addition, recent proteomic and genetic studies have revealed the presence of distinct BRCA1 complexes in vivo, each of which governs a specific cellular response to DNA damage. Thus, BRCA1 is emerging as the master regulator of the genome through its ability to execute and coordinate various aspects of the DNA damage response. © 2010 Macmillan Publishers Limited. All rights reserved.link_to_subscribed_fulltex

Regulation of Chromatin Architecture by the PWWP Domain-Containing DNA Damage-Responsive Factor EXPAND1/MUM1

Dynamic changes of chromatin structure facilitate diverse biological events, including DNA replication, repair, recombination, and gene transcription. Recent evidence revealed that DNA damage elicits alterations to the chromatin to facilitate proper checkpoint activation and DNA repair. Here we report the identification of the PWWP domain-containing protein EXPAND1/MUM1 as an architectural component of the chromatin, which in response to DNA damage serves as an accessory factor to promote cell survival. Depletion of EXPAND1/MUM1 or inactivation of its PWWP domain resulted in chromatin compaction. Upon DNA damage, EXPAND1/MUM1 rapidly concentrates at the vicinity of DNA damage sites via its direct interaction with 53BP1. Ablation of this interaction impaired damage-induced chromatin decondensation, which is accompanied by sustained DNA damage and hypersensitivity to genotoxic stress. Collectively, our study uncovers a chromatin-bound factor that serves an accessory role in coupling damage signaling with chromatin changes in response to DNA damage. © 2010 Elsevier Inc. All rights reserved.link_to_subscribed_fulltex

Specific recognition of phosphorylated tail of H2AX by the tandem BRCT domains of MCPH1 revealed by complex structure

MCPH1 is especially important for linking chromatin remodeling to DNA damage response. It contains three BRCT (BRCA1-carboxyl terminal) domains. The N-terminal region directly binds with chromatin remodeling complex SWI-SNF, and the C-terminal BRCT2-BRCT3 domains (tandem BRCT domains) are involved in cellular DNA damage response. The MCPH1 gene associates with evolution of brain size, and its variation can cause primary microcephaly. In this study we solve the crystal structures of MCPH1 natural variant (A761) C-terminal tandem BRCT domains alone as well as in complex with γH2AX tail. Compared with other structures of tandem BRCT domains, the most significant differences lie in phosphopeptide binding pocket. Additionally, fluorescence polarization assays demonstrate that MCPH1 tandem BRCT domains show a binding selectivity on pSer +3 and prefer to bind phosphopeptide with free COOH-terminus. Taken together, our research provides new structural insights into BRCT-phosphopeptide recognition mechanism. © 2011 Elsevier Inc.link_to_subscribed_fulltex

The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA

Individuals with BRCA2 mutations are predisposed to breast cancers owing to genome instability. To determine the functions of BRCA2, the human protein was purified. It was found to bind selectively to single-stranded DNA (ssDNA), and to ssDNA in tailed duplexes and replication fork structures. Monomeric and dimeric forms of BRCA2 were observed by EM. BRCA2 directed the binding of RAD51 recombinase to ssDNA, reduced the binding of RAD51 to duplex DNA and stimulated RAD51-mediated DNA strand exchange. These observations provide a molecular basis for the role of BRCA2 in the maintenance of genome stability