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Role of the catalytic metal during polymerization by DNA polymerase lambda

Authors
Journal
DNA Repair
1568-7864
Publisher
Elsevier
Publication Date
Volume
6
Issue
9
Identifiers
DOI: 10.1016/j.dnarep.2007.03.005
Keywords
  • Dna Polymerase Lambda
  • Dna Repair
  • Nucleotidyl Transfer
  • Phosphoryl Transfer
  • X-Ray Crystallography
  • Crystal Structure
  • Divalent Metal
  • Catalysis
  • Manganese
  • Non-Hydrolyzable Nucleotide
Disciplines
  • Biology

Abstract

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 λ (Pol λ), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol λ. In a 1.9 Å structure of Pol λ 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 α-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl 2 yielded a 2.0 Å structure with Mn 2+ occupying the site for metal A. In the presence of Mn 2+, 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 α-phosphate (3.69 Å) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl 2 converted a pre-catalytic Pol λ/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.

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