Maintenance of genomic stability ensures faithful transmission of genetic information and helps suppress neoplastic transformation and tumorigenesis. Although recent progress has advanced our understanding of DNA damage checkpoint regulations, little is known as to how DNA repair, especially the RAD51-dependent homologous recombination repair pathway, is executed in vivo. Here, we reveal novel properties of the BRCA2-associated protein PALB2 in the assembly of the recombinational DNA repair machinery at DNA damage sites. Although the chromatin association of PALB2 is a prerequisite for subsequent BRCA2 and RAD51 loading, the focal accumulation of the PALB2⅐BRCA2⅐RAD51 complex at DSBs occurs independently of known DNA damage checkpoint and repair proteins. We provide evidence to support that PALB2 exists as homo-oligomers and that PALB2 oligomerization is essential for its focal accumulation at DNA breaks in vivo. We propose that both PALB2 chromatin association and its oligomerization serve to secure the BRCA2⅐RAD51 repair machinery at the sites of DNA damage. These attributes of PALB2 are likely instrumental for proficient homologous recombination DNA repair in the cell.Fanconi anemia is a rare disease in which patients are prone to the development of childhood aplastic anemia and cancer as well as other congenital defects. Cellular phenotypes of FA 4 patients are also characterized by their hypersensitivity toward DNA-cross-linking agents, such as mitomycin C (MMC) or cisplatin. Accordingly, MMC treatment greatly induces aberrant chromosomal structures in cells derived from FA patients, including chromosome breakage and chromatin interchanges. Thus, genomic instability is considered as one of the fundamental causes responsible for the clinical and cellular phenotypes observed among FA patients.In human cells two major repair pathways are employed to repair DSBs, namely the homologous recombination (HR) and the non-homologous end-joining pathways. The use of the sister chromatid as information donor during repair renders HR a largely faithful mechanism (1), whereas non-homologous endjoining often leads to genetic mutations because of the gain or loss of genetic information (2).Mounting evidence suggests a functional connection between the 13 FA-complementation group genes (FA-A, B/FAAP95, C, D1/BRCA2, D2, E, F, G/XRCC9, I, J/BACH1, L/PHF9/FAAP43, M/Hef/FAAP250, and N/PALB2) and the DNA repair pathway (3). Recent studies revealed that eight of the FA proteins form a complex to facilitate the ubiquitylation of FANCD2 and FANCI; however, mechanistically how they affect DNA repair remains elusive. Importantly, the identification of the FANCJ/BACH1, FANCD1/BRCA2, and FANCN/ PALB2 proteins as components of the HR machinery further support the notion that FA mutations result in DNA repair defects (3-7).Genetics and biochemical studies have shown that the FANCD1 product, BRCA2, facilitates the assembly of RAD51 onto ssDNA substrates, forming a nucleoprotein filament (8 -10) that catalyzes DNA strand invasion and D-loop...

PALB2 is an integral component of the BRCA complex important for recombinational DNA repair. However, exactly how this activity is regulated in vivo remains unexplored. Here we provide evidence to show that MRG15 is a novel PALB2-associated protein that ensures regulated recombination events. We found that the direct interaction between MRG15 and PALB2 is mediated by an evolutionarily conserved region on PALB2. Intriguingly, although damage-induced RAD51 foci formation and mitomycin C sensitivity appeared normal in MRG15-binding defective PALB2 mutants, these cells exhibited a significant increase in gene conversion rates. Consistently, we found that abrogation of the PALB2-MRG15 interaction resulted in elevated sister chromatid exchange frequencies. Our results suggest that loss of the PALB2-MRG15 interaction relieved the cells with the suppression of sister chromatid exchange and therefore led to a hyper-recombination phenotype in the gene conversion assay. Together, our study indicated that although PALB2 is required for proficient homologous recombination, it could also govern the choice of templates used in homologous recombination repair.

The Fanconi anemia (FA) pathway participates in interstrand crosslink (ICL) repair and the maintenance of genomic stability. The FA core complex consists of eight FA proteins and two Fanconi anemiaassociated proteins (FAAP24 and FAAP100). The FA core complex has ubiquitin ligase activity responsible for monoubiquitination of the FANCI-FANCD2 (ID) complex, which in turn initiates a cascade of biochemical events that allow processing and removal of crosslinked DNA and thereby promotes cell survival following DNA damage. Here, we report the identification of a unique component of the FA core complex, namely, FAAP20, which contains a RAD18-like ubiquitin-binding zinc-finger domain. Our data suggest that FAAP20 promotes the functional integrity of the FA core complex via its direct interaction with the FA gene product, FANCA. Indeed, somatic knockout cells devoid of FAAP20 displayed the hallmarks of FA cells, including hypersensitivity to DNA cross-linking agents, chromosome aberrations, and reduced FANCD2 monoubiquitination. Taking these data together, our study indicates that FAAP20 is an important player involved in the FA pathway.is a rare recessive genetic disorder characterized by bone marrow failure, congenital developmental defects, and cancer predisposition (1-4). Cellular features of FA include chromosomal instability and hypersensitivity to crosslinking agents (5). Fifteen FA complementation genes have been identified so far. These genes form several complexes to orchestrate interstrand cross-linking (ICL) repair. The FA core complex is composed of eight of the FA gene products (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM), in addition to FAAP24 and FAAP100 (6, 7), and acts as an E3 ligase to ubiquitinate FANCI/FANCD2 (I/D2) complex (8-11). The monoubiquitinated FANCI/FANCD2 complex interacts with Fanconi anemia-associated nuclease 1 (FAN1), which has exonuclease and endonuclease activity that may unhook the ICL, facilitate translesion synthesis, and promote downstream homologous recombination (HR) repair (12-15).Besides the FA core complex and FANCI/FANCD2, there are several other FA proteins that likely act downstream of FANCI/ FANCD2 and participate in HR repair. These proteins include BRCA2 (FANCD1) and PALB2 (FANCN), both of which are essential for HR repair (16-18). Another downstream FA protein, BACH1 (FANCJ), is also a bona fide double-strand break repair factor (19). BRCA2/FANCD1, PALB2/FANCN, and BACH1/FANCJ are all recruited to ICL sites (20), indicating that they are directly involved in ICL repair. More recently, mutations in two other genes, RAD51C/FANCO and SLX4/FANCP, were identified in patients with FA phenotypes (21-23), suggesting that there may be additional FA genes responsible for this disease.Not all FA proteins function in a linear pathway involved in ICL repair. Many of the downstream FA proteins are involved in HR repair and associated with breast cancer susceptibility (16-19, 24, 25). These proteins all have functions besides ICL repair. More recently, another DN...

Western blottingCell lysates were prepared and Western blots were performed using standard protocols. Phospho-ATM antibodies were obtained from Rockland, and Chk1 and phospho-Chk1 antibodies were purchased from Cell Signaling Technology. Anti -mouse ATM antibodies were generously provided by Y. Shiloh (Tel Aviv University, Tel Aviv, Israel).The G2/M checkpoint assay Cells were irradiated (2 Gy) and were harvested 1 h later. Cells were then fi xed and stained with antiphospho-H3 antibodies (Cell Signaling Technology). Mitotic population (mitotic index) was determined by FACS analysis. Fig. S1 shows that DKO MEFs do not have more severe defects in the ATMdependent DNA damage signaling pathway. Online supplemental material is available at Online supplemental material

Ubc13 is an E2 ubiquitin conjugating enzyme that functions in nuclear DNA damage signaling and cytoplasmic NF-κB signaling. Here we present the structures of complexes of Ubc13 with two inhibitors, NSC697923 and BAY 11-7082, which inhibit DNA damage and NF-κB signaling in human cells. NSC697923 and BAY 11-7082 both inhibit Ubc13 by covalent adduct formation through a Michael addition at the Ubc13 active site cysteine. The resulting adducts of both compounds exploit a binding groove unique to Ubc13. We developed a Ubc13 mutant which resists NSC697923 inhibition and, using this mutant, we show that the inhibition of cellular DNA damage and NF-κB signaling by NSC697923 is largely due to specific Ubc13 inhibition. We propose that unique structural features near the Ubc13 active site could provide a basis for the rational development and design of specific Ubc13 inhibitors.