title: Role of the catalytic metal during polymerization by DNA polymerase lambda.
abstract: The incorporation of dNMPs into DNA by polymerases involves a phosphoryl transfer reaction hypothesized to require two divalent metal ions. Here we investigate this hypothesis using as a model human DNA polymerase lambda (Pol lambda), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol lambda. In a 1.9 A structure of Pol lambda containing a 3'-OH and the non-hydrolyzable analog dUpnpp, a non-catalytic Na+ ion occupies the site for metal A and the ribose of the primer-terminal nucleotide is found in a conformation that positions the acceptor 3'-OH out of line with the alpha-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl2 yielded a 2.0 A structure with Mn2+ occupying the site for metal A. In the presence of Mn2+, the conformation of the ribose is C3'-endo and the 3'-oxygen is in line with the leaving oxygen, at a distance from the phosphorus atom of the alpha-phosphate (3.69 A) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl2 converted a pre-catalytic Pol lambda/Na+ complex with unreacted dCTP in the active site into a product complex via catalysis in the crystal. These data provide pre- and post-transition state information and outline in a single crystal the pathway for the phosphoryl transfer reaction carried out by DNA polymerases.
authors.affiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
authors.forename: Miguel
authors.initials: M
authors.lastname: Garcia-Diaz
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Bebenek
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Krahn
authors.forename: Lars C
authors.initials: LC
authors.lastname: Pedersen
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kunkel
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008345:Manganese / Q000494:pharmacology*; D008958:Models, Molecular; D016296:Mutagenesis; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D010710:Phosphates; D008345:Manganese; D004247:DNA; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta