Mechanistic insights into RAD51-associated protein 1 (RAD51AP1) action in homologous DNA repair

Homologous recombination catalyzed by the RAD51 recombinase is essential for maintaining genome integrity upon the induction of DNA double strand breaks and other DNA lesions. By enhancing the recombinase activity of RAD51, RAD51AP1 (RAD51-associated protein 1) serves a key role in homologous recombination-mediated chromosome damage repair. We show here that RAD51AP1 harbors two distinct DNA binding domains that are both needed for maximal protein activity under physiological conditions. We have finely mapped the two DNA binding domains in RAD51AP1 and generated mutant variants that are impaired in either or both of the DNA binding domains. Examination of these mutants reveals that both domains are indispensable for RAD51AP1 function in cells. These and other results illuminate the mechanistic basis of RAD51AP1 action in homologous DNA repair.     

RAD51-associated protein 1 (RAD51AP1) interacts with the meiotic recombinase DMC1 through a conserved motif

Homologous recombination (HR) reactions mediated by the RAD51 recombinase are essential for DNA and replication fork repair, genome stability, and tumor suppression. RAD51-associated protein 1 (RAD51AP1) is an important HR factor that associates with and stimulates the recombinase activity of RAD51. We have recently shown that RAD51AP1 also partners with the meiotic recombinase DMC1, displaying isoform-specific interactions with DMC1. Here, we have characterized the DMC1 interaction site in RAD51AP1 by a series of truncations and point mutations to uncover a highly conserved WVPP motif critical for DMC1 interaction but dispensable for RAD51 association. This RAD51AP1 motif is reminiscent of the FVPP motif in the tumor suppressor protein BRCA2 that mediates DMC1 interaction. These results further implicate RAD51AP1 in meiotic HR via RAD51 and DMC1.     

Distinct roles of FANCO/RAD51C protein in DNA damage signaling and repair: implications for Fanconi anemia and breast cancer susceptibility

RAD51C, a RAD51 paralog, has been implicated in homologous recombination (HR), and germ line mutations in RAD51C are known to cause Fanconi anemia (FA)-like disorder and breast and ovarian cancers. The role of RAD51C in the FA pathway of DNA interstrand cross-link (ICL) repair and as a tumor suppressor is obscure. Here, we report that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G(2)/M accumulation, which are hallmarks of the FA phenotype. We find that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrate that RAD51C plays a vital role in the HR-mediated repair of DNA lesions associated with replication. Finally, we show that RAD51C participates in ICL and double strand break-induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C that were identified in FA and breast and ovarian cancers reveal that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor.     

The unstructured C-terminal tail of yeast Dpb11 (human TopBP1) protein is dispensable for DNA replication and the S phase checkpoint but required for the G2/M checkpoint

Budding yeast Dpb11 (human TopBP1, fission yeast Cut5) is an essential protein required for replisome assembly and for the DNA damage checkpoint. Previous studies with the temperature-sensitive dpb11-1 allele, truncated at amino acid 583 of the 764-amino acid protein, have suggested the model that Dpb11 couples DNA replication to the replication checkpoint. However, the dpb11-1 allele shows distinct replication defects even at permissive temperatures. Here, we determine that the 1-600-amino acid domain of DPB11 is both required and sufficient for full replication function of Dpb11 but that this domain is defective for activation of the principal checkpoint kinase Mec1 (human ataxia telangiectasia and Rad3-related) in vitro and in vivo. Remarkably, mutants of DPB11 that leave its replication function intact but abrogate its ability to activate Mec1 are proficient for the replication checkpoint, but they are compromised for the G(2)/M DNA damage checkpoint. These data suggest that replication checkpoint defects may result indirectly from defects in replisome assembly. Two conserved aromatic amino acids in the C terminus of Dpb11 are critical for Mec1 activation in vitro and for the G(2)/M checkpoint in yeast. Together with aromatic motifs identified previously in the Ddc1 subunit of 9-1-1, another activator of Mec1 kinase, they define a consensus structure for Mec1 activation.