Efficiency of Correct Nucleotide Insertion Governs DNA Polymerase Fidelity

DNA polymerase fidelity or specificity expresses the ability of a polymerase to select a correct nucleoside triphosphate (dNTP) from a pool of structurally similar molecules. Fidelity is quantified from the ratio of specificity constants (catalytic efficiencies) for alternate substrates (i.e. correct and incorrect dNTPs). An analysis of the efficiency of dNTP (correct and incorrect) insertion for a low fidelity mutant of DNA polymerase β (R283A) and exonuclease-deficient DNA polymerases from five families derived from a variety of biological sources reveals that a strong correlation exists between the ability to synthesize DNA and the probability that the polymerase will make a mistake (i.e.base substitution error). Unexpectedly, this analysis indicates that the difference between low and high fidelity DNA polymerases is related to the efficiency of correct, but not incorrect, nucleotide insertion. In contrast to the loss of fidelity observed with the catalytically inefficient R283A mutant, the fidelity of another inefficient mutant of DNA polymerase β (G274P) is not altered. Thus, although all natural low fidelity DNA polymerases are inefficient, not every inefficient DNA polymerase has low fidelity. Low fidelity polymerases appear to be an evolutionary solution to how to replicate damaged DNA or DNA repair intermediates without burdening the genome with excessive polymerase-initiated errors. DNA polymerase fidelity or specificity expresses the ability of a polymerase to select a correct nucleoside triphosphate (dNTP) from a pool of structurally similar molecules. Fidelity is quantified from the ratio of specificity constants (catalytic efficiencies) for alternate substrates (i.e. correct and incorrect dNTPs). An analysis of the efficiency of dNTP (correct and incorrect) insertion for a low fidelity mutant of DNA polymerase β (R283A) and exonuclease-deficient DNA polymerases from five families derived from a variety of biological sources reveals that a strong correlation exists between the ability to synthesize DNA and the probability that the polymerase will make a mistake (i.e.base substitution error). Unexpectedly, this analysis indicates that the difference between low and high fidelity DNA polymerases is related to the efficiency of correct, but not incorrect, nucleotide insertion. In contrast to the loss of fidelity observed with the catalytically inefficient R283A mutant, the fidelity of another inefficient mutant of DNA polymerase β (G274P) is not altered. Thus, although all natural low fidelity DNA polymerases are inefficient, not every inefficient DNA polymerase has low fidelity. Low fidelity polymerases appear to be an evolutionary solution to how to replicate damaged DNA or DNA repair intermediates without burdening the genome with excessive polymerase-initiated errors. The equilibrium between genome stability and instability is tightly regulated since mutations are central to aging, disease, and evolution. Thus, cellular strategies that modulate this equilibrium are of general and immense interest. The structure of DNA was proposed nearly 50 years ago and provided the first clue to how “genetic material” could be replicated faithfully (1Watson J.D. Crick F.H.C. Nature. 1953; 171: 737-738Google Scholar). It is now recognized that DNA polymerases play pivotal roles in both genome replication and maintenance (i.e. DNA repair). Polymerases copy the parental (template) strand to generate a new or repaired complementary daughter strand, and accurate DNA synthesis during replication and repair is essential in maintaining genomic integrity. Although DNA polymerases play a central role in these essential processes, the fundamental mechanism(s) by which they select the correct deoxynucleoside 5′-triphosphate (dNTP) 1The abbreviations used are: dNTP, 2′-deoxynucleoside 5′-triphosphate; pol, polymerase from a pool of structurally similar molecules to accomplish efficient and faithful polymerization is poorly understood. The intrinsic base substitution error frequency for DNA replication and repair polymerases is generally between 10−3 and 10−6 (2Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Google Scholar). These frequencies represent one error per thousand or million nucleotides synthesized, respectively. These levels of discrimination are far greater than predicted by free energy differences between matched and mismatched DNA termini (predicted error frequency of ∼0.4; one error per 3 nucleotides synthesized), indicating that DNA polymerases can enhance fidelity by a large factor (3Petruska J. Goodman M.F. Boosalis M.S. Sowers L.C. Cheong C. Tonoco J. Ignacio Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6252-6256Google Scholar). However, even this remarkable specificity is inadequate to faithfully replicate a genome of more than 109 nucleotides. Thus, replicative DNA polymerases often have an intrinsic proofreading exonuclease to remove misinserted nucleotides, and cells possess a postreplication DNA mismatch repair pathway that can correct misinserted nucleotides that escape proofreading. DNA polymerase (pol) fidelity, specificity, or discrimination represent relative kinetic terms used to describe the propensity of a polymerase to produce a base substitution error. Polymerase specificity may be quantified in vitro by measuring the insertion kinetics of a single nucleotide (correct or incorrect) opposite a defined templating base. The absolute rate or probability that a pol inserts a correct or incorrect nucleotide follows Michaelis-Menten kinetics. A steady-state kinetic approach defines substrate specificity as catalytic efficiency,k cat/K m,dNTP, for formation of a specific base pair. Substrate specificity determined by a pre-steady-state kinetic approach isk pol/K d whereK d is the equilibrium dissociation constant for the incoming dNTP and k pol is the kinetic step that limits insertion of the first nucleotide. This step may be either chemistry (i.e. nucleotidyl tran