title: Y265H mutator mutant of DNA polymerase beta. Proper teometric alignment is critical for fidelity.
abstract: DNA polymerases have the unique ability to select a specific deoxynucleoside triphosphate from a pool of similarly structured substrates. One of these enzymes, DNA polymerase beta, offers a simple system to relate polymerase structure to the fidelity of DNA synthesis. In this study, a mutator DNA polymerase beta, Y265H, was identified using an in vivo genetic screen. Purified Y265H produced errors at a 40-fold higher frequency than the wild-type protein in a forward mutation assay. At 37 degrees C, transient kinetic analysis demonstrated that the alteration caused a 111-fold decrease in the maximum rate of polymerization and a 117-fold loss in fidelity for G misincorporation opposite template A. Our data suggest that the maximum rate of polymerization was reduced, because Y265H was dramatically impaired in its ability to perform nucleotidyl transfer in the presence of the correct nucleotide substrate. In contrast, at 20 degrees C, the mutant protein had a fidelity similar to wild-type enzyme. Both proteins at 20 degrees C demonstrate a rapid change in protein conformation, followed by a slow chemical step. These data suggest that proper geometric alignment of template, 3'-OH of the primer, magnesium ions, dNTP substrates, and the active site residues of DNA polymerase beta are important factors in polymerase fidelity and provide the first evidence that Tyr-265 is important for this alignment to occur properly in DNA polymerase beta.
authors.affiliation: Departments of Therapeutic Radiology, Genetics, and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520.
authors.forename: A M
authors.initials: AM
authors.lastname: Shah
authors.forename: S X
authors.initials: SX
authors.lastname: Li
authors.forename: K S
authors.initials: KS
authors.lastname: Anderson
authors.forename: J B
authors.initials: JB
authors.lastname: Sweasy
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics*; D009154:Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D019951:DNA Polymerase beta