Kinetic effect of a downstream strand and its 5'-terminal moieties on single nucleotide gap-filling synthesis catalyzed by human DNA polymerase lambda

J Biol Chem. 2006 Nov 24;281(47):35649-55. doi: 10.1074/jbc.M607479200. Epub 2006 Sep 27.


During short-patch base excision repair, the excision of a 5'-terminal 2-deoxyribose-5-phosphate moiety of the downstream strand by the 5'-2-deoxyribose-5-phosphate lyase activity of either DNA polymerase beta or lambda is believed to occur after each respective enzyme catalyzes gap-filling DNA synthesis. Yet the effects of this 5'-terminal 2-deoxyribose-5-phosphate moiety on the polymerase activities of these two enzymes have never been quantitatively determined. Moreover, x-ray crystal structures of truncated polymerase lambda have revealed that the downstream strand and its 5'-phosphate group of gapped DNA interact intensely with the dRPase domain, but the kinetic effect of these interactions is unclear. Here, we utilized pre-steady state kinetic methods to systematically investigate the effect of a downstream strand and its 5'-moieties on the polymerase activity of the full-length human polymerase lambda. The downstream strand and its 5'-phosphate were both found to increase nucleotide incorporation efficiency (kp/Kd) by 15 and 11-fold, respectively, with the increase procured by the effect on the nucleotide incorporation rate constant kp rather than the ground state nucleotide binding affinity Kd. With 4 single nucleotide-gapped DNA substrates containing a 1,2-dideoxyribose-5-phosphate moiety, a 2-deoxyribose-5-phosphate mimic, we measured the incorporation efficiencies of 16 possible nucleotides. Our results demonstrate that although this 5'-terminal 2-deoxyribose-5-phosphate mimic does not affect the fidelity of polymerase lambda, it moderately decreased the polymerase efficiency by 3.4-fold. Moreover, this decrease in polymerase efficiency is due to a drop of similar magnitude in kp rather than Kd. The implication of the downstream strand and its 5'-moieties on the kinetics of gap-filling synthesis is discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Crystallography, X-Ray
  • DNA Polymerase beta / chemistry*
  • DNA Polymerase beta / metabolism
  • Dose-Response Relationship, Drug
  • Humans
  • In Situ Nick-End Labeling
  • Kinetics
  • Models, Chemical
  • Molecular Sequence Data
  • Nucleotides / chemistry
  • Oligonucleotides / chemistry
  • Protein Binding
  • Protein Structure, Tertiary
  • Temperature


  • Nucleotides
  • Oligonucleotides
  • DNA polymerase beta2
  • DNA Polymerase beta