title: Loss of DNA polymerase beta stacking interactions with templating purines, but not pyrimidines, alters catalytic efficiency and fidelity.
abstract: Structures of DNA polymerases bound with DNA reveal that the 5'-trajectory of the template strand is dramatically altered as it exits the polymerase active site. This distortion provides the polymerase access to the nascent base pair to interrogate proper Watson-Crick geometry. Upon binding a correct deoxynucleoside triphosphate, alpha-helix N of DNA polymerase beta is observed to form one face of the binding pocket for the new base pair. Asp-276 and Lys-280 stack with the bases of the incoming nucleotide and template, respectively. To determine the role of Lys-280, site-directed mutants were constructed at this position, and the proteins were expressed and purified, and their catalytic efficiency and fidelity were assessed. The catalytic efficiency for single-nucleotide gap filling with the glycine mutant (K280G) was strongly diminished relative to wild type for templating purines (>15-fold) due to a decreased binding affinity for the incoming nucleotide. In contrast, catalytic efficiency was hardly affected by glycine substitution for templating pyrimidines (<4-fold). The fidelity of the glycine mutant was identical to the wild type enzyme for misinsertion opposite a template thymidine, whereas the fidelity of misinsertion opposite a template guanine was modestly altered. The nature of the Lys-280 side-chain substitution for thymidine triphosphate insertion (templating adenine) indicates that Lys-280 "stabilizes" templating purines through van der Waals interactions.
authors.affiliation: Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
authors.forename: William A
authors.initials: WA
authors.lastname: Beard
authors.forename: David D
authors.initials: DD
authors.lastname: Shock
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Yang
authors.forename: Saundra F
authors.initials: SF
authors.lastname: DeLauder
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
mesh_terms: D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D020137:Base Pair Mismatch; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011687:Purines / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*; D013942:Thymine Nucleotides / Q000378:metabolism
chemical_list: D011687:Purines; D011743:Pyrimidines; D013942:Thymine Nucleotides; D001224:Aspartic Acid; D004247:DNA; D001120:Arginine; D019951:DNA Polymerase beta; D008239:Lysine; C024157:thymidine 5'-triphosphate; D005998:Glycine