Human DNA polymerase N (PolN) is an A-family nuclear DNA polymerase whose function is unknown. This study examines the possible role of PolN in DNA repair in human cells treated with PolN-targeted siRNA. HeLa cells with siRNA-mediated knockdown of PolN were more sensitive than control cells to DNA cross-linking agent mitomycin C (MMC), but were not hyper-sensitive to UV irradiation. The MMC hyper-sensitivity of PolN knockdown cells was rescued by the overexpression of DNA polymerase-proficient PolN but not by DNA polymerase-deficient PolN. Furthermore, in vitro experiments showed that purified PolN conducts low efficiency non-mutagenic bypass of a psoralen DNA interstrand cross-link (ICL), whose structure resembles an intermediate in the proposed pathway of ICL repair. These results suggest that PolN might play a role in translesion DNA synthesis during ICL repair in human cells. KeywordsDNA interstrand cross-links; DNA polymerase N; TLS; Psoralen; Chemotherapeutics E. coli PolI is a high fidelity DNA repair polymerase that conducts gap-filling DNA synthesis during nucleotide excision repair (NER), base excision repair (BER), and DNA interstrand cross-link (ICL) repair. E. coli PolI is the prototypical member of A-family DNA polymerases (1). Drosophila melanogaster Mus308 is an A-family nuclear DNA polymerase, the mutants of which are hyper-sensitive to DNA cross-linking agents but not to other DNA damaging agents (2)(3)(4). This suggests that Mus308 may play a role in ICL repair in Drosophila. Two nuclear A-family DNA polymerases, DNA polymerase N (PolN) and DNA polymerase Q (PolQ) were recently discovered (5,6). It has been proposed that PolN and PolQ are mammalian orthologs of Mus308 and that they participate in ICL repair in mammalian cells. However, additional studies are needed to confirm the precise role(s) of PolN and PolQ in DNA repair in mammalian cells.ICLs are generated endogenously as bi-functional products of lipid peroxidation and by exogenous exposure to DNA damaging agents, some of which are commonly used as cancer chemotherapeutic drugs (7,8). Because ICLs covalently link the two complementary strands of duplex DNA, they prevent progression of the DNA replication fork and block RNA transcription. This property makes ICLs highly toxic to proliferating cells. The molecular mechanism of human ICL repair is poorly understood (7-10). The current model of mammalian ICL repair (7-10) suggests that when a DNA replication fork stalls at an ICL, it is recognized *To whom correspondence should be addressed. tbessho@unmc by an endonuclease that generates a double-strand break (DSB) in the vicinity of the ICL (11)(12)(13)(14). Subsequently, XPF-ERCC1 unhooks the ICL, resulting in a gap across from the unhooked ICL (14-17), which cannot be sealed by replicative DNA polymerases δ or ε. Gaps generated during ICL repair are thought to be repaired by translesion DNA synthesis (TLS) or by homologous recombination using the homologous chromosome (the sister chromatid is not available). Once the gap is sea...
DNA interstrand cross-links (ICLs) are mainly repaired by the combined action of nucleotide excision repair and homologous recombination in E. coli. Genetic data also suggest the existence of a nucleotide excision repair-dependent, homologous recombination-independent ICL repair pathway. The involvement of translesion synthesis in this pathway has been postulated; however, the molecular mechanism of this pathway is not understood. To examine the role of translesion synthesis in ICL repair, we generated a defined substrate with a single psoralen ICL that mimics a postincision structure generated by nucleotide excision repair. We demonstrated that the Klenow fragment (DNA polymerase I) performs translesion synthesis on this model substrate. This in vitro translesion synthesis assay will help in understanding the basic mechanism of a postincision translesion synthesis process in ICL repair.
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