title: Analysis of the impact of a uracil DNA glycosylase attenuated in AP-DNA binding in maintenance of the genomic integrity in Escherichia coli.
abstract: 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.
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Sanjay Kumar
authors.initials: SK
authors.lastname: Bharti
authors.forename: Umesh
authors.initials: U
authors.lastname: Varshney
mesh_terms: 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*
chemical_list: D029968:Escherichia coli Proteins; C540081:rpoB protein, E coli; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D051981:Uracil-DNA Glycosidase