The Activity of Natural Polymorphic Variants of Human DNA Polymerase β Having an Amino Acid Substitution in the Transferase Domain

Abstract

To maintain the integrity of the genome, there is a set of enzymatic systems, one of which is base excision repair (BER), which includes sequential action of DNA glycosylases, apurinic/apyrimidinic endonucleases, DNA polymerases, and DNA ligases. Normally, BER works efficiently, but the enzymes themselves (whose primary function is the recognition and removal of damaged bases) are subject to amino acid substitutions owing to natural single-nucleotide polymorphisms (SNPs). One of the enzymes in BER is DNA polymerase β (Polβ), whose function is to fill gaps in DNA with complementary dNMPs. It is known that many SNPs can cause an amino acid substitution in this enzyme and a significant decrease in the enzymatic activity. In this study, the activity of four natural variants of Polβ, containing substitution E154A, G189D, M236T, or R254I in the transferase domain, was analyzed using molecular dynamics simulations and pre-steady-state kinetic analyses. It was shown that all tested substitutions lead to a significant reduction in the ability to form a complex with DNA and with incoming dNTP. The G189D substitution also diminished Polβ catalytic activity. Thus, a decrease in the activity of studied mutant forms may be associated with an increased risk of damage to the genome.

SNP-Associated Substitutions of Amino Acid Residues in the dNTP Selection Subdomain Decrease Polβ Polymerase Activity

“…Many known amino acid substitutions generated by SNPs weaken polymerase activity of Polβ during the filling of a 1-nt gap and during extension of the primer strand [27,32,55,57]. To investigate the influence of amino acid substitutions G274R, G290C, and R333W on the polymerase activity of the enzyme, products of the enzymatic reaction were separated by polyacrylamide gel (PAAG) electrophoresis.…”

“…The detected change in the polymerase activity of the Polβ variants G274R, G290C, and R333W may be explained not only by the shift of affinity for 1-nt gap-containing DNA but also by a change in the ability to bind complementary dNTPs. For many polymorphic variants of Polβ, there is evidence of a decrease in observed dissociation constant K d,app(dATP) , as well as a decrease in polymerization constant k pol (reflecting the rate of the chemical stage) [32,55].…”

The Impact of SNP-Induced Amino Acid Substitutions L19P and G66R in the dRP-Lyase Domain of Human DNA Polymerase β on Enzyme Activities

“…It has been shown previously that a 2-aminopurine (2-aPu) fluorescent residue is a sensitive label that could help with the detection of both binding of dNTP and its incorporation into a DNA substrate [53][54][55][56]. Moreover, stopped-flow analysis of Polβ interaction with DNA and dNTPs has been well characterized by means of 2-aPu fluorescence intensity changes [41,42,57,58]. It has been stated that two-stage changes in fluorescence intensity of a 2-aPu residue correspond to (i) the stage of formation of a ternary closed complex of the enzyme, DNA, and dNTP (an increase phase) and to (ii) the chemical stage of transfer of the dNMP residue to the 3 ′ end of a primer and formation of the reaction product (a phase of a decrease in fluorescence intensity) [57][58][59][60].…”