title: Insights into the role of Val45 and Gln182 of Escherichia coli MutY in DNA substrate binding and specificity.
abstract: BACKGROUND
  Escherichia coli MutY (EcMutY) reduces mutagenesis by removing adenines paired with guanines or 7,8-dihydro-8-oxo-guanines (8-oxoG). V45 and Q182 of EcMutY are considered to be the key determinants of adenine specificity. Both residues are spatially close to each other in the active site and are conserved in MutY family proteins but not in Methanobacterium thermoautotrophicum Mig.MthI T/G mismatch DNA glycosylase (A50 and L187 at the corresponding respective positions).
  
  
  RESULTS
  Targeted mutagenesis study was performed to determine the substrate specificities of V45A, Q182L, and V45A/Q182L double mutant of EcMutY. All three mutants had significantly lower binding and glycosylase activities for A/G and A/8-oxoG mismatches than the wild-type enzyme. The double mutant exhibited an additive reduction in binding to both the A/G and A/GO in comparison to the single mutants. These mutants were also tested for binding and glycosylase activities with T/G- or T/8-oxoG-containing DNA. Both V45A and Q182L mutants had substantially increased affinities towards T/G, however, they did not exhibit any T/G or T/8-oxoG glycosylase activity. Surprisingly, the V45A/Q182L double mutant had similar binding affinities to T/G as the wild-type EcMutY. V45A, Q182L, and V45A/Q182L EcMutY mutants could not reduce the G:C to T:A mutation frequency of a mutY mutant. Expression of the V45A mutant protein caused a dominant negative phenotype with an increased G:C to A:T mutation frequency.
  
  
  CONCLUSIONS
  The substrate specificities are altered in V45A, Q182L, and V45A/Q182L EcMutY mutants. V45A and Q182L mutants had reduced binding and glycosylase activities for A/G and A/8-oxoG mismatches and increased affinities towards T/G mismatch. However, in contrast to a previous report that Mig.MthI thymine DNA glycosylase can be converted to a MutY-like adenine glycosylase by replacing two residues (A50V and L187Q), both V45A and Q182L EcMutY mutants did not exhibit any T/G or T/8-oxoG glycosylase activity. The dominant negative phenotype of V45A EcMutY mutant protein is probably caused by its increased binding affinity to T/G mismatch and thus inhibiting other repair pathways.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, 108 North Greene Street, Baltimore, MD 21201, USA. andy_chang_drat@yahoo.co.jp
authors.forename: Po-Wen
authors.initials: PW
authors.lastname: Chang
authors.forename: Amrita
authors.initials: A
authors.lastname: Madabushi
authors.forename: A-Lien
authors.initials: AL
authors.lastname: Lu
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D045647:DNA Glycosylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053843:DNA Mismatch Repair; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011485:Protein Binding; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D045647:DNA Glycosylases; C061738:mutY adenine glycosylase