Hinge residue I174 Is critical for proper dNTP selection by DNA polymerase

Jen Yamtich, Daniela Starcevic, Julia Lauper, Elenoe Smith, Idina Shi, Sneha Rangarajan, Joachim Jaeger, Joann B. Sweasy

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

DNA polymerase β (pol. β) is the key gap-filling polymerase in base excision, repair, the DNA repair pathway responsible for repairing up to 20000 endogenous lesions per cell per day. Pol. β is also widely used as a model polymerase for structure and function studies, and several, structural regions have been identified as being critical, for the fidelity of the enzyme. One of these regions is the hydrophobic hinge, a network of hydrophobic residues located between the palm and fingers subdomains. Previous work by our lab has shown that hinge residues Y265, I260, and F272 are critical for polymerase fidelity by functioning in discrimination of the correct from incorrect dNTP during ground state binding. Our work aimed to elucidate the role of hinge residue I174 in polymerase fidelity. To study this residue, we conducted, a genetic screen to identify mutants with a substitution at residue I174 that resulted in a mutator polymerase. We then chose the mutator mutant I174S for further study and found that it follows the same general kinetic pathway as and has an overall protein folding similar to that of wild-type (WT) pol β. Using single-turnover kinetic analysis, we found that I174S exhibits decreased fidelity when inserting a nucleotide opposite a template base G, and this loss of fidelity is due primarily to a loss of discrimination during ground state dNTP binding. Molecular dynamics simulations show that mutation of residue I174 to serine results in an overall tightening of the hinge region, resulting in aberrant protein dynamics and fidelity. These results point to the hinge region as being critical in the maintenance of the proper geometry of the dNTP binding pocket.

Original languageEnglish (US)
Pages (from-to)2326-2334
Number of pages9
JournalBiochemistry
Volume49
Issue number11
DOIs
StatePublished - Mar 23 2010
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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