Analysis of the impact of a uracil DNA glycosylase attenuated in AP-DNA binding in maintenance of the genomic integrity in Escherichia coli

Bharti, Sanjay Kumar and Varshney, Umesh (2010) Analysis of the impact of a uracil DNA glycosylase attenuated in AP-DNA binding in maintenance of the genomic integrity in Escherichia coli. In: Nucleic Acids Research, 38 (7). pp. 2291-2301.

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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 similar to 7- and similar to 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 similar to 5-, similar to 3.0- and similar to 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.

Item Type:	Journal Article
Publication:	Nucleic Acids Research
Publisher:	Oxford University Press
Additional Information:	Copyright of this article belongs to Oxford University Press.
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	07 Jun 2010 09:07
Last Modified:	19 Sep 2010 06:01
URI:	http://eprints.iisc.ac.in/id/eprint/27438

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