Structures of DNA polymerase beta with active-site mismatches suggest a transient abasic site intermediate during misincorporation.
We report the crystallographic structures of DNA polymerase beta with dG-dAMPCPP and dC-dAMPCPP mismatches in the active site. These premutagenic structures were obtained with a nonhydrolyzable incoming nucleotide analog, dAMPCPP, and Mn(2+). Substituting Mn(2+) for Mg(2+) significantly decreases the fidelity of DNA synthesis. The structures reveal that the enzyme is in a closed conformation like that observed with a matched Watson-Crick base pair. The incorrect dAMPCPP binds in a conformation identical to that observed with the correct nucleotide. To accommodate the incorrect nucleotide and closed protein conformation, the template strand in the vicinity of the active site has shifted upstream over 3 A, removing the coding base from the active site and generating an abasic templating pocket. The primer terminus rotates as its complementary template base is repositioned. This rotation moves O3' of the primer terminus away from the alpha-phosphate of the incoming nucleotide, thereby deterring misincorporation.
Batra
Vinod K
VK
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709, USA.
Beard
William A
WA
Shock
David D
DD
Pedersen
Lars C
LC
Wilson
Samuel H
SH
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D020137:Base Pair Mismatch; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation
D008345:Manganese; D000255:Adenosine Triphosphate; D004247:DNA; D019951:DNA Polymerase beta; C002630:alpha,beta-methyleneadenosine 5'-triphosphate