title: Structural mechanism of ribonucleotide discrimination by a Y-family DNA polymerase.
abstract: The ability of DNA polymerases to differentiate between ribonucleotides and deoxribonucleotides is fundamental to the accurate replication and maintenance of an organism's genome. The active sites of Y-family DNA polymerases are highly solvent accessible, yet these enzymes still maintain a high selectivity towards deoxyribonucleotides. Here, we biochemically demonstrate that a single active-site mutation (Y12A) in Dpo4, a model Y-family DNA polymerase, causes both a dramatic loss of ribonucleotide discrimination and a decrease in nucleotide incorporation efficiency. We also determined two ternary crystal structures of the Dpo4 Y12A mutant incorporating either dATP or ATP nucleotides opposite a template dT base. Interestingly, both dATP and ATP were hydrolyzed to dADP and ADP, respectively. In addition, the dADP and ADP molecules adopt a similar conformation and position at the polymerase active site to a ddADP molecule in the ternary crystal structure of wild-type Dpo4. The Y12A mutant loses stacking interactions with the deoxyribose of dNTP, which destabilizes the binding of incoming nucleotides. The mutation also opens a space to accommodate the 2'-OH group of the ribose of NTP in the polymerase active site. The structural change leads to the reduction in deoxynucleotide incorporation efficiency and allows ribonucleotide incorporation.
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
authors.forename: Kevin N
authors.initials: KN
authors.lastname: Kirouac
authors.forename: Zucai
authors.initials: Z
authors.lastname: Suo
authors.forename: Hong
authors.initials: H
authors.lastname: Ling
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D012265:Ribonucleotides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003854:Deoxyribonucleotides; D012265:Ribonucleotides; D004259:DNA-Directed DNA Polymerase