Involvement of human DNA polymerase kappa in nucleotide excision repair

Nucleotide excision repair is one of the major repair pathways responsible for identifying and removing lesions in the DNA double helix. In higher eukaryotes, nucleotide excision repair is a coordinated response of over 30 proteins recruited in an ordered procession with distinct roles in the recognition, removal and repair of said lesions. A key step in the completion of the repair process is the resynthesis of the excised strand using the undamaged partner as a template. DNA polymerase kappa (polκ), a member of the Y-family, has been shown to have a role in nucleotide excision repair distinct from its traditional role in translesion synthesis. Cell lines lacking polκ showed clear defects in nucleotide excision repair and increased ultraviolet light sensitivity. Building on this established work, conserved residues were identified in the C-terminus of human polκ and mutated to alanines. Under transient expression, mutations in the ubiquitin binding domains severely impaired the recruitment to sites of damage. Cell lines defective in polκ that stably expressed these mutant polymerases showed sensitivity to ultraviolet radiation following exposure; intriguingly, this defect seems confined to the global genomic repair pathway as no substantial defect in transcription-coupled repair was observed. Following on from these observations, immunoprecipitation of the polymerase and partner proteins was investigated in an attempt to identify interactions disrupted by the mutations to the ubiquitin binding domains. These experiments indicated impairment in binding to ubiquitinated PCNA in the mutants. In further work, the recruitment of wild-type human polκ was shown to be independent of the 3' incision by the nuclease XPG during the repair process, consistent with a recently proposed model for NER

BAF180 promotes cohesion and prevents genome instability and aneuploidy

BAF180, a subunit of the PBAF chromatin remodeling complex, is frequently mutated in cancer. Although PBAF regulates transcription, it remains unclear whether this is what drives tumorigenesis in cells lacking BAF180. Based on data from yeast, we hypothesized that BAF180 may prevent tumorigenesis by promoting cohesion. Here, we show BAF180 is required for centromeric cohesion in mouse and human cells. Mutations identified in tumor samples are unable to support this activity, and also compromise cohesion-dependent functions in yeast. We provide evidence of genome instability in line with loss of cohesion, and importantly, we find dynamic chromosome instability following DNA damage in cells lacking BAF180. These data demonstrate a function for BAF180 in promoting genome stability that is distinct from its well-characterized role in transcriptional regulation, uncovering a potent mechanism for its tumor-suppressor activity

Three DNA polymerases, recruited by different mechanisms, carry out NER repair synthesis in human cells

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both polκ and polδ, and both polymerases can be recovered in the same repair complexes. Polκ is recruited to repair sites by ubiquitinated PCNA and XRCC1 and polδ by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on polɛ, recruitment of which is dependent on the alternative clamp loader CTF18-RFC