A unified kinetic mechanism applicable to multiple DNA polymerases.
After extensive studies spanning over half a century, there is little consensus on the kinetic mechanism of DNA polymerases. Using stopped-flow fluorescence assays for mammalian DNA polymerase beta (Pol beta), we have previously identified a fast fluorescence transition corresponding to conformational closing, and a slow fluorescence transition matching the rate of single-nucleotide incorporation. Here, by varying pH and buffer viscosity, we have decoupled the rate of single-nucleotide incorporation from the rate of the slow fluorescence transition, thus confirming our previous hypothesis that this transition represents a conformational event after chemistry, likely subdomain reopening. Analysis of an R258A mutant indicates that rotation of the Arg258 side chain is not rate-limiting in the overall kinetic pathway of Pol beta, yet is kinetically significant in subdomain reopening. We have extended our kinetic analyses to a high-fidelity polymerase, Klenow fragment (KF), and a low-fidelity polymerase, African swine fever virus DNA polymerase X (Pol X), and showed that they follow the same kinetic mechanism as Pol beta, while differing in relative rates of single-nucleotide incorporation and the putative conformational reopening. Our data suggest that the kinetic mechanism of Pol beta is not an exception among polymerases, and furthermore, its delineated kinetic mechanism lends itself as a platform for comparison of the kinetic properties of different DNA polymerases and their mutants.
Bakhtina
Marina
M
Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Roettger
Michelle P
MP
Kumar
Sandeep
S
Tsai
Ming-Daw
MD
D000358:African Swine Fever Virus / Q000201:enzymology; D000409:Alanine / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D002384:Catalysis; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats
D001120:Arginine; C109534:DNA polymerase X; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D000409:Alanine