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Catalytic contributions of key residues in the adenine glycosylase MutY revealed by pH-dependent kinetics and cellular repair assays.

ABSTRACT: MutY prevent DNA mutations associated with 8-oxoguanine (OG) by catalyzing the removal of adenines opposite OG. pH dependence of the adenine glycosylase activity establish that Asp 138 of MutY must be deprotonated for maximal activity consistent with its role in stabilizing the oxacarbenium ion transition state in an S(N)1 mechanism. A cellular OG:A repair assay allowed further validation of the critical role of Asp 138. Conservative substitutions of the catalytic residues Asp 138 and Glu 37 resulted in enzymes with a range of activity that were used to correlate the efficiency of adenine excision with overall OG:A repair and suppression of DNA mutations in vivo. The results show that MutY variations that exhibit reduced mismatch affinity result in more dramatic reductions in cellular OG:A repair than those that only compromise adenine excision catalysis.

SUBMITTER: Brinkmeyer MK

PROVIDER: S-EPMC3292766 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Catalytic contributions of key residues in the adenine glycosylase MutY revealed by pH-dependent kinetics and cellular repair assays.

Brinkmeyer Megan K MK Pope Mary Ann MA David Sheila S SS

Chemistry & biology 20120201 2

MutY prevent DNA mutations associated with 8-oxoguanine (OG) by catalyzing the removal of adenines opposite OG. pH dependence of the adenine glycosylase activity establish that Asp 138 of MutY must be deprotonated for maximal activity consistent with its role in stabilizing the oxacarbenium ion transition state in an S(N)1 mechanism. A cellular OG:A repair assay allowed further validation of the critical role of Asp 138. Conservative substitutions of the catalytic residues Asp 138 and Glu 37 res ...[more]

PMID: 22365610

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Project description:Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian Uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect, unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprised of the cognate viral DNA polymerase, vPOL encoded by ORF9 , and the viral DNA polymerase processivity factor, vPPF encoded by ORF59 . MHV68 vUNG colocalized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone, or in combination. Last, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity.ImportanceGammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo . In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus to form a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus associated cancers.

Dataset Information

Catalytic contributions of key residues in the adenine glycosylase MutY revealed by pH-dependent kinetics and cellular repair assays.

Publications

Catalytic contributions of key residues in the adenine glycosylase MutY revealed by pH-dependent kinetics and cellular repair assays.

Similar Datasets

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