Analysis of the impact of a uracil DNA glycosylase attenuated in AP-DNA binding in maintenance of the genomic integrity in Escherichia coli.
Uracil DNA glycosylase (Ung) initiates the uracil excision repair pathway. We have earlier characterized the Y66W and Y66H mutants of Ung and shown that they are compromised by approximately 7- and approximately 170-fold, respectively in their uracil excision activities. In this study, fluorescence anisotropy measurements show that compared with the wild-type, the Y66W protein is moderately compromised and attenuated in binding to AP-DNA. Allelic exchange of ung in Escherichia coli with ung::kan, ungY66H:amp or ungY66W:amp alleles showed approximately 5-, approximately 3.0- and approximately 2.0-fold, respectively increase in mutation frequencies. Analysis of mutations in the rifampicin resistance determining region of rpoB revealed that the Y66W allele resulted in an increase in A to G (or T to C) mutations. However, the increase in A to G mutations was mitigated upon expression of wild-type Ung from a plasmid borne gene. Biochemical and computational analyses showed that the Y66W mutant maintains strict specificity for uracil excision from DNA. Interestingly, a strain deficient in AP-endonucleases also showed an increase in A to G mutations. We discuss these findings in the context of a proposal that the residency of DNA glycosylase(s) onto the AP-sites they generate shields them until recruitment of AP-endonucleases for further repair.
Bharti
Sanjay Kumar
SK
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Varshney
Umesh
U
D000483:Alleles; D019943:Amino Acid Substitution; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D016680:Genome, Bacterial; D009154:Mutation; D051981:Uracil-DNA Glycosidase / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; C540081:rpoB protein, E coli; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D051981:Uracil-DNA Glycosidase