Determinants of nucleotide sugar recognition in an archaeon DNA polymerase

Abstract

Vent DNA polymerase normally discriminates strongly against incorporation of ribonucleotides, 3'-deoxyribonucleotides (such as cordycepin) and 2',3'-dideoxyribonucleotides. To explore the basis for this discrimination we have generated a family of variants with point mutations of residues in conserved Regions II and III and assayed incorporation of nucleo-tides with modified sugars by these variants, all of which were created in an exonuclease-deficient form of the enzyme. A Y412V variant incorporates ribonucleotides at least 200-fold more efficiently than the wild-type enzyme, consistent with Y412 acting as a 'steric gate' to specifically exclude ribonucleotides. The most striking variants tested involved changes to A488, a residue predicted to be facing away from the nucleotide binding site. The pattern of relaxed specificity at this position roughly correlates with the size of the substituted amino acid sidechain and affects a variety of modified nucleotide sugars.

Comparative Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase

“…Analysis of Enhanced Nucleotide Analog Incorporation by Vent A488L DNA Polymerase-We previously reported enhanced incorporation of nucleotide analogs by Vent A488L DNA polymerase (32,33). In a burst kinetics experiment, the A488L mutant enzyme gave an initial burst of dCTP incorporation at a rate similar to that seen with the wild-type enzyme (k burst ϭ 45 s Ϫ1 ; Ͼ75% active) (Table I).…”

“…Nucleotides, Nucleotide Analogs, DNA Substrate, and Enzymes-All DNA polymerases used in this study are 3Ј 3 5Ј exonuclease-deficient as a result of mutation of catalytic aspartic and glutamic acids to alanine in the exonuclease active site (31,32,45). These mutations prevent exonuclease removal of newly incorporated nucleotides or terminators.…”

“…1). Furthermore, steadystate kinetic studies have identified hyperthermophilic DNA polymerase residues important for polymerization and exonuclease activities and for nucleotide binding (18,29,(31)(32)(33)(34)(35). Nucleotide analogs have also been important in identifying dNTP recognition determinants important in the polymerase reaction (32-36) and have proven useful in a variety of molecular biology applications, such as DNA sequencing and detection of single nucleotide polymorphisms (37-41).…”

“…Furthermore, steadystate kinetic studies have identified hyperthermophilic DNA polymerase residues important for polymerization and exonuclease activities and for nucleotide binding (18,29,(31)(32)(33)(34)(35). Nucleotide analogs have also been important in identifying dNTP recognition determinants important in the polymerase reaction (32)(33)(34)(35)(36) and have proven useful in a variety of molecular biology applications, such as DNA sequencing and detection of single nucleotide polymorphisms (37)(38)(39)(40)(41). One group of analogs, 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs), 1 is particularly intriguing due to the wide spectrum of incorporation efficiency noted in different DNA polymerases, even within the same family of polymerase.…”

“…In the course of probing the determinants of nucleotide sugar discrimination in the Family B DNA polymerase from the hyperthermophilic Archaea Thermococcus litoralis (Vent DNA polymerase), we identified a mutant (Vent A488L DNA polymerase) that reduces discrimination against several altered nucleotides (32,33). Subsequent crystal structures of closely related DNA polymerases strongly suggested that this residue makes neither direct nor indirect contacts with the reaction substrates, raising questions about the structural basis for the observed variation (Fig.…”