DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 A. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress.

Mutations in breast cancer susceptibility gene 1 and 2 (BRCA1 and BRCA2) predispose individuals to breast and ovarian cancer development. We previously reported an in vivo interaction between BRCA1 and BRCA2. However, the biological significance of their association is thus far undefined. Here, we report that PALB2, the partner and localizer of BRCA2, binds directly to BRCA1, and serves as the molecular scaffold in the formation of the BRCA1-PALB2-BRCA2 complex. The association between BRCA1 and PALB2 is primarily mediated via apolar bonding between their respective coiled-coil domains. More importantly, BRCA1 mutations identified in cancer patients disrupted the specific interaction between BRCA1 and PALB2. Consistent with the converging functions of the BRCA proteins in DNA repair, cells harboring mutations with abrogated BRCA1-PALB2 interaction resulted in defective homologous recombination (HR) repair. We propose that, via its direct interaction with PALB2, BRCA1 fine-tunes recombinational repair partly through its modulatory role in the PALB2-dependent loading of BRCA2-RAD51 repair machinery at DNA breaks. Our findings uncover PALB2 as the molecular adaptor between the BRCA proteins, and suggest that impaired HR repair is one of the fundamental causes for genomic instability and tumorigenesis observed in patients carrying BRCA1, BRCA2, or PALB2 mutations. Breast cancer and ovarian cancer is estimated to be responsible for more than one-fifth of cancer mortality (1). Because germ-line mutations in breast cancer susceptibility gene 1 and 2 (BRCA1 and BRCA2) account for the development of a significant portion of hereditary breast and ovarian cancer, the understanding of their roles in tumor suppression is crucial to the improvement of therapeutic interventions. Because accumulation of genetic aberrations are often observed in cells derived from familial breast cancer patients with BRCA1 or BRCA2 mutations, the BRCA proteins have been considered as the caretakers of genomic integrity. Accordingly, tumor cells derived from these patients exhibit hypersensitivity to DNA damaging agents and display genomic instability (2-4).Given the similar phenotypes in BRCA1 and BRCA2 patients, and the spectrum of deficits observed in cells deficient in these proteins, one would envision that the BRCA proteins might work in synchrony in certain cellular process(es) essential for tumor suppression. Consistent with this notion, interaction between the 2 BRCA proteins has been reported (5). However, exactly how their interaction is regulated and the biological significance for such interaction remains largely unexplored.BRCA1 participates in numerous cellular processes (3, 6-8). In particular, BRCA1 has been proposed to have diverse roles to promote cell survival in response to genotoxic stress. Recent elucidation of multiple BRCA1 complexes in vivo suggests a multifactorial model by which BRCA1 mediates distinct processes that include checkpoint activation, damage signaling, and DNA repair (9, 10). However, BRCA2 has a pivotal r...

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.

To maintain genome stability, cells respond to DNA damage by activating signaling pathways that govern cell cycle checkpoints and initiate DNA repair. Cell cycle checkpoint controls should somehow connect with DNA repair processes, however, exactly how such coordination occurs in vivo is largely unknown. Here we revealed a novel role of RAD18 as the integral component that translates the damage response signal to orchestrate homologous recombination (HR) repair. We show that RAD18 promotes HR in a manner strictly dependent upon its ability to be recruited to the sites of DNA breaks and this recruitment relies on a well-defined DNA damage-signaling pathway mediated by another E3 ligase RNF8. We further demonstrate that RAD18 functions as an adaptor to facilitate HR via a direct interaction with RAD51C. Together, our data uncovers RAD18 as a key factor that orchestrates HR repair via surveillance of the DNA damage signal.

npg Post-translational modifications play a crucial role in coordinating cellular response to DNA damage. Recent evidence suggests an interplay between multiple protein modifications, including phosphorylation, ubiquitylation, acetylation and sumoylation, that combine to propagate the DNA damage signal to elicit cell cycle arrest, DNA repair, apoptosis and senescence. Utility of specific post-translational modifiers allows temporal and spatial control over protein relocalization and interactions, and may represent a means for trans-regulatory activation of protein activities. The ability to recognize these specific modifiers also underscores the capacity for signal amplification, a crucial step for the maintenance of genomic stability and tumor prevention. Here we have summarized recent findings that highlight the complexity of post-translational modifications in coordinating the DNA damage response, with emphasis on the DNA damage signaling cascade.