title: Hinge residue I174 is critical for proper dNTP selection by DNA polymerase beta.
abstract: DNA polymerase beta (pol beta) 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 beta 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 beta. 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.
authors.affiliation: Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Jen
authors.initials: J
authors.lastname: Yamtich
authors.forename: Daniela
authors.initials: D
authors.lastname: Starcevic
authors.forename: Julia
authors.initials: J
authors.lastname: Lauper
authors.forename: Elenoe
authors.initials: E
authors.lastname: Smith
authors.forename: Idina
authors.initials: I
authors.lastname: Shi
authors.forename: Sneha
authors.initials: S
authors.lastname: Rangarajan
authors.forename: Joachim
authors.initials: J
authors.lastname: Jaeger
authors.forename: Joann B
authors.initials: JB
authors.lastname: Sweasy
mesh_terms: D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007532:Isoleucine; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D051381:Rats; D013379:Substrate Specificity
chemical_list: D009711:Nucleotides; D007532:Isoleucine; D004247:DNA; D019951:DNA Polymerase beta