We have previously described

an in vitro system that allo

We have previously described

an in vitro system that allows us to measure mutation and transformation frequencies in H. pylori wild type strains and isogenic gene knock-out mutants, as well as the length of the donor DNA fragments imported into the recipient chromosome after transformation [12]. In this system, natural transformation of different H. pylori wild type strains with DNA from heterologous H. pylori donors led to the incorporation of 1.3-3.8 kb fragments into the recipient chromosome, depending on the combination Selleck Caspase inhibitor of donor and recipient strains. Imports resulting from recombination contained short interspersed sequences of the recipient (ISR) in ~10% of the cases [12, 13], leading to complex mosaic patterns. The glycosylase MutY, a member of the base excision repair (BER) machinery, is involved in at least one ISR-generating pathway in H. pylori, repairing mismatches after the heteroduplex formation between recipient and donor DNA [12]. However, the inactivation of mutY in H. pylori did not Selleck CT99021 completely abrogate the formation of ISR, suggesting that additional mechanisms might contribute to ISR generation. In addition to BER, H. pylori also contains a second gap-filling DNA repair system, the nucleotide excision repair pathway (NER), whose role in H. pylori mutation and PD0332991 recombination is yet poorly understood. In Escherichia coli, the NER

system is responsible for the replacement of bulky DNA lesions such as covalently modified bases, noncovalent drug nucleotide complexes and abasic sites generated by oxidative metabolism or ionizing radiation [14, 15]. Initiation of NER starts with the recognition of DNA distortions by the UvrAB complex [16]. After recognition, UvrA dissociates and UvrC is recruited and acts as a single-stranded DNA endonuclease, cleaving at both sides of the lesion CYTH4 [17, 18]. Finally, the unwinding activity of the UvrD helicase, which preferentially catalyzes a 3’ to

5’ unwinding, removes the excised segment. DNA polymerase I fills in the gap while the remaining nick is closed by ligase [19, 20]. In H. pylori, orthologs of the four NER genes, uvrA-D, have been identified [21]; but until now, only few studies have addressed the functions of these genes. H. pylori UvrB was shown to be involved in the repair of acid-induced DNA damage [22], and UvrD limited homologous recombination and DNA damage-induced genomic rearrangements between DNA repeats [23]. Here we have used a genetic approach to analyze the roles of the H. pylori NER system components in regulating the mutation rate, and the frequency and import patterns of homologous recombination after natural transformation. Results Characterization of H. pylori NER mutants and their susceptibility to UV light-induced cell damage To investigate how the NER system contributes to genetic diversification in H. pylori, we individually inactivated the NER genes in H.

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