Comparative Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase

Abstract

Comparative kinetic and structural analyses of a variety of polymerases have revealed both common and divergent elements of nucleotide discrimination. Although the parameters for dNTP incorporation by the hyperthermophilic archaeal Family B Vent DNA polymerase are similar to those previously derived for Family A and B DNA polymerases, parameters for analog incorporation reveal alternative strategies for discrimination by this enzyme. Discrimination against ribonucleotides was characterized by a decrease in the affinity of NTP binding and a lower rate of phosphoryl transfer, whereas discrimination against ddNTPs was almost exclusively due to a slower rate of phosphodiester bond formation. Unlike Family A DNA polymerases, incorporation of 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs) by Vent DNA polymerase was enhanced over ddNTPs via a 50-fold increase in phosphoryl transfer rate. Furthermore, a mutant with increased propensity for nucleotide analog incorporation (Vent A488L DNA polymerase) had unaltered dNTP incorporation while displaying enhanced nucleotide analog binding affinity and rates of phosphoryl transfer. Based on kinetic data and available structural information from other DNA polymerases, we propose active site models for dNTP, ddNTP, and acyNTP selection by hyperthermophilic archaeal DNA polymerases to rationalize structural and functional differences between polymerases.All free living organisms encode several DNA polymerases that are jointly responsible for the replication and maintenance of their genomes, thereby ensuring accurate transmission of genetic information (1-3). The majority of identified DNA polymerases can be classified into Families A, B, C, and Y according to amino acid sequence similarities to Escherichia coli polymerases I, II, III, and IV/V, respectively (4, 5). Additional families have been identified, including the two-subunit replicative DNA polymerases from hyperthermophilic Archaea (Family D) (6) and eukaryotic DNA polymerase ␤ and terminal transferases (Family X) (4).Structural and kinetic analyses of Family A (7-14) and Family B (15-25) DNA polymerases have increased the understanding of nucleotide selection and incorporation mechanisms. Although amino acid sequences diverge between these two families, the structures of Family A and B DNA polymerases share recognizable finger, thumb, and palm subdomains that allow comparison of structural elements important for function (3, 11). In the case of Family A DNA polymerases from bacteriophage T7, Escherichia coli (Klenow fragment, large fragment of DNA polymerase I), and Thermus aquaticus, as well as the Family B DNA polymerase from bacteriophage RB69, interpretation of the structural information is complemented by steady-state and pre-steady-state kinetic studies, allowing a detailed description of the polymerization pathway. Reaction parameters describing the discrimination against naturally occurring nucleotide analogs encountered in vivo, such as NTPs, or unnatural n...

DNA Polymerase β Ribonucleotide Discrimination

“…possessing a 2Ј-OH) and select 2Ј-deoxyribose triphosphates. Previous studies with A-and B-family polymerases have found significant effects on nucleotide incorporation as a result of modifying the deoxyribose ring (3)(4)(5)(6)(7)(8). DNA polymerases insert ribonucleotides with a much lower efficiency than deoxynucleotides with the same base due to a slower rate of insertion and weaker binding.…”

“…We modified the upstream template sugar from dGMP to rGMP and annealed the resulting template to the dC-and rC-terminated primers to create rG-dC and rG-rC (template-primer) 5 termini, respectively. We maintained correct Watson-Crick base pairing at the terminus in all sequence contexts because mismatched termini have been shown to have variable effects on dNTP extension (15).…”

Enzymatic Incorporation of an Antibody‐Activated Blue Fluorophore into DNA

“…[11] Notably, the overall fidelity of Vent exo À DNA polymerase is still twofold higher than that of Taq DNA polymerase. To underscore the significance of our findings, we sought to compare the incorporation data of compound 9 with pyrene-8-dATP, a structurally related, commercially available, nonnatural fluorescent nucleotide in its role as substrate for the four DNA polymerases tested.…”

Archaeal DNA polymerases in biotechnology

“…9°N-7 DNA pol mutants can also effectively incorporate modified nucleotides in addition to ddNMP. Dye-acyNTP terminators can be incorporated by the A485L 9°N-7 DNA pol exo-mutant (Therminator™) for DNA sequencing and provides useful sequence beyond 1000 nucleotides (Gardner et al 2004). Furthermore, Therminator™ (D141A/E143A/A485L), Therminator™ II (D141A/ E143A/Y409V/A485L), and Therminator™ III (D141A/ E143A/L408S/Y409A/ P410V) can incorporate acyclo chain terminators for SNP analysis.…”