RESUMO
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 hypersensitive to UV irradiation. The MMC hypersensitivity 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 nonmutagenic 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.
Assuntos
Dano ao DNA , Enzimas Reparadoras do DNA/fisiologia , Reparo do DNA , DNA Polimerase Dirigida por DNA/fisiologia , Sequência de Bases , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Núcleo Celular/genética , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA/genética , DNA Helicases/química , DNA Helicases/fisiologia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/biossíntese , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/deficiência , DNA Polimerase Dirigida por DNA/genética , Células HeLa , Humanos , Mitomicina/farmacologia , Dados de Sequência Molecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , RNA Interferente Pequeno/farmacologiaRESUMO
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.
