DNA polymerase beta: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes.
Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase beta (Pol beta) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (kpol) and the apparent equilibrium dissociation constant of dNTP (Kd) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates. For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the Kd of dNTP. Although kpol was little affected, the catalytic efficiency kpol/Kd increased significantly owing to the decreases in Kd. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the Kd of dNTP appreciably but caused the kpol (and thus kpol/Kd) for incorrect dNTP incorporation to increase. As a consequence the fidelity of Pol beta was not significantly affected by the use of single-nucleotide gapped DNA substrates. In addition we show that under processive polymerization conditions the processivity of Pol beta increases in the gap-filling synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed. However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected. A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol beta already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.
Ahn
J
J
Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, USA.
Kraynov
V S
VS
Zhong
X
X
Werneburg
B G
BG
Tsai
M D
MD
D004247:DNA / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D004926:Escherichia coli; D005453:Fluorescence; D007700:Kinetics; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D013379:Substrate Specificity
D003854:Deoxyribonucleotides; D004247:DNA; D019951:DNA Polymerase beta