Human Polβ Natural Polymorphic Variants G118V and R149I Affects Substate Binding and Catalysis

Abstract

DNA polymerase β (Polβ) expression is essential for the cell’s response to DNA damage that occurs during natural cellular processes. Polβ is considered the main reparative DNA polymerase, whose role is to fill the DNA gaps arising in the base excision repair pathway. Mutations in Polβ can lead to cancer, neurodegenerative diseases, or premature aging. Many single-nucleotide polymorphisms have been identified in the POLB gene, but the consequences of these polymorphisms are not always clear. It is known that some polymorphic variants in the Polβ sequence reduce the efficiency of DNA repair, thereby raising the frequency of mutations in the genome. In the current work, we studied two polymorphic variants (G118V and R149I separately) of human Polβ that affect its DNA-binding region. It was found that each amino acid substitution alters Polβ’s affinity for gapped DNA. Each polymorphic variant also weakens its binding affinity for dATP. The G118V variant was found to greatly affect Polβ’s ability to fill gapped DNA and slowed the catalytic rate as compared to the wild-type enzyme. Thus, these polymorphic variants seem to decrease the ability of Polβ to maintain base excision repair efficiency.

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

“…The collected protein fraction was dialyzed overnight against a buffer composed of, 20 mM HEPES-KOH pH 7.8, 150 mM NaCl and 20% glycerol. The pure protein fraction was supplemented with 50% glycerol and stored at −20 • C. For more details, refer to [32].…”

“…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].…”

For the Better or for the Worse? The Effect of Manganese on the Activity of Eukaryotic DNA Polymerases

“…Finally, the remaining nick is sealed by a DNA ligase. Both the 5′-dRP lyase and polymerase activities are important for genomic integrity, as cancer-associated mutations exclusively affecting either the polymerase or the lyase domain were identified [ 28 , 32 , 33 ]. Moreover, Polβ is mutated in ~30% of human tumors, and disruption of the POLB gene coding for Polβ in mice results in embryonic lethality, which emphasizes the important role of the enzyme in maintaining genome stability [ 34 , 35 , 36 ].…”

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

“…To date, many polymorphic variants with reduced enzymatic characteristics have been described for DNA polymerase β [22][23][24][25][26]. Moreover, up to 30% of analyzed human tumors have been shown to express Polβ mutants [27].…”