Role of the catalytic metal during polymerization by DNA polymerase lambda

DNA Repair (Amst). 2007 Sep 1;6(9):1333-40. doi: 10.1016/j.dnarep.2007.03.005. Epub 2007 May 1.

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.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Binding Sites
  • Catalysis
  • Crystallization
  • Crystallography, X-Ray
  • DNA / chemistry*
  • DNA / metabolism*
  • DNA Polymerase beta / chemistry*
  • DNA Polymerase beta / genetics
  • DNA Polymerase beta / metabolism*
  • Humans
  • Manganese / pharmacology*
  • Models, Molecular
  • Mutagenesis*
  • Phosphates / metabolism
  • Protein Binding
  • Protein Conformation

Substances

  • Phosphates
  • Manganese
  • DNA
  • DNA polymerase beta2
  • DNA Polymerase beta