Hinge residue Ile260 of DNA polymerase beta is important for enzyme activity and fidelity.
DNA polymerases ensure efficient insertion of the correct dNTP into the DNA substrate. They have evolved mechanisms for discriminating among very similar dNTP substrates. DNA polymerase beta is a repair polymerase that provides a model system for a direct study of insertion fidelity. In this study, we examined the role of hinge residue Ile260 of the rat Polbeta on enzyme activity and accuracy. We changed residue I260 to every other amino acid residue and used genetic screens to assess the activity and fidelity of the resulting mutants. The I260D, -E, -K, -N, and -R mutants are significantly less active than wild-type Polbeta. Interestingly, I260H and I260Q are active but exhibit mutator activity. This suggests that the nonpolar nature of residue 260 is important for maintaining the activity and fidelity of Polbeta. We employ molecular modeling as an aid in explaining the observed phenotypes and propose a mechanism whereby the positioning of the DNA substrate in the enzyme and within the surface of the hinge may be a key player in forming an optimal active site for phosphodiester bond formation between Watson-Crick base pairs.
Starcevic
Daniela
D
Program in Microbiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Dalal
Shibani
S
Sweasy
Joann
J
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D014644:Genetic Variation; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D013379:Substrate Specificity / Q000235:genetics
D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D007532:Isoleucine; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D001216:Asparagine; D001120:Arginine; D019951:DNA Polymerase beta; D008239:Lysine