Bharti, SK and Varshney, U (2010) Analysis of the impact of a uracil DNA glycosylase attenuated in AP-DNA binding in maintenance of the genomic integrity in Escherichia coli. Nucleic Acids Res. 38:2291-301
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.
Alleles; Amino Acid Substitution; DNA/metabolism; DNA Damage; DNA Repair; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Genome, Bacterial; Mutation; Uracil-DNA Glycosidase/genetics; Uracil-DNA Glycosidase/metabolism
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