Biomaterial Database

A unified kinetic mechanism applicable to multiple DNA polymerases. 2007

Marina Bakhtina, and Michelle P Roettger, and Sandeep Kumar, and Ming-Daw Tsai

Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.

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.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D011487	Protein Conformation	The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).	Conformation, Protein,Conformations, Protein,Protein Conformations
D002384	Catalysis	The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction.	Catalyses
D004256	DNA Polymerase I	A DNA-dependent DNA polymerase characterized in prokaryotes and may be present in higher organisms. It has both 3'-5' and 5'-3' exonuclease activity, but cannot use native double-stranded DNA as template-primer. It is not inhibited by sulfhydryl reagents and is active in both DNA synthesis and repair.	DNA Polymerase alpha,DNA-Dependent DNA Polymerase I,Klenow Fragment,DNA Pol I,DNA Dependent DNA Polymerase I,Polymerase alpha, DNA
D004259	DNA-Directed DNA Polymerase	DNA-dependent DNA polymerases found in bacteria, animal and plant cells. During the replication process, these enzymes catalyze the addition of deoxyribonucleotide residues to the end of a DNA strand in the presence of DNA as template-primer. They also possess exonuclease activity and therefore function in DNA repair.	DNA Polymerase,DNA Polymerases,DNA-Dependent DNA Polymerases,DNA Polymerase N3,DNA Dependent DNA Polymerases,DNA Directed DNA Polymerase,DNA Polymerase, DNA-Directed,DNA Polymerases, DNA-Dependent,Polymerase N3, DNA,Polymerase, DNA,Polymerase, DNA-Directed DNA,Polymerases, DNA,Polymerases, DNA-Dependent DNA
D000358	African Swine Fever Virus	The lone species of the genus Asfivirus. It infects domestic and wild pigs, warthogs, and bushpigs. Disease is endemic in domestic swine in many African countries and Sardinia. Soft ticks of the genus Ornithodoros are also infected and act as vectors.	Wart-Hog Disease Virus,Virus, Wart-Hog Disease,Wart Hog Disease Virus
D000409	Alanine	A non-essential amino acid that occurs in high levels in its free state in plasma. It is produced from pyruvate by transamination. It is involved in sugar and acid metabolism, increases IMMUNITY, and provides energy for muscle tissue, BRAIN, and the CENTRAL NERVOUS SYSTEM.	Abufène,Alanine, L-Isomer,L-Alanine,Alanine, L Isomer,L Alanine,L-Isomer Alanine
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D001120	Arginine	An essential amino acid that is physiologically active in the L-form.	Arginine Hydrochloride,Arginine, L-Isomer,DL-Arginine Acetate, Monohydrate,L-Arginine,Arginine, L Isomer,DL Arginine Acetate, Monohydrate,Hydrochloride, Arginine,L Arginine,L-Isomer Arginine,Monohydrate DL-Arginine Acetate
D017434	Protein Structure, Tertiary	The level of protein structure in which combinations of secondary protein structures (ALPHA HELICES; BETA SHEETS; loop regions, and AMINO ACID MOTIFS) pack together to form folded shapes. Disulfide bridges between cysteines in two different parts of the polypeptide chain along with other interactions between the chains play a role in the formation and stabilization of tertiary structure.	Tertiary Protein Structure,Protein Structures, Tertiary,Tertiary Protein Structures