Kinetic effect of a downstream strand and its 5'-terminal moieties on single nucleotide gap-filling synthesis catalyzed by human DNA polymerase lambda.
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.
Duym
Wade W
WW
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Fiala
Kevin A
KA
Bhatt
Nikunj
N
Suo
Zucai
Z
D001483:Base Sequence; D018360:Crystallography, X-Ray; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020287:In Situ Nick-End Labeling; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013696:Temperature
D009711:Nucleotides; D009841:Oligonucleotides; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta