Critical amino acids in human DNA polymerases eta and kappa involved in erroneous incorporation of oxidized nucleotides

Abstract

Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human Poleta and Polkappa proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Poleta was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type Poleta) to 7 : 1 (R61K). Similarly, Tyr112 in Polkappa was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Polkappa) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Poleta, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Poleta and Polkappa might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.

Figure 1.

Incorporation of oxidized dNTPs into DNA by Polκ and its Y112A mutant. The primer/template DNA (100 nM, N in the template strand represents A, C, G or T) was incubated with wild-type Polκ (10 nM) or Y112A mutant (100 nM) in the presence of 50 µM 8-oxo-dGTP (A), 8-oxo-dATP (B) or 2-OH-dATP (C) for 10 min at 37°C. Extended primers were separated by denaturing PAGE. The first lanes represent the results of control experiments where no dNTPs were added to the reaction mixtures.

Figure 2.

Incorporation of oxidized dNTPs into DNA by Polη and its R61K mutant. The primer/template DNA (100 nM, N in the template strand represents A, C, G or T) was incubated with wild-type Polη and R61K mutant (5 nM) in the presence of 50 µM 8-oxo-dGTP (A), 8-oxo-dATP (B) or 2-OH-dATP (C) for 10 min at 37°C. Extended primers were separated by denaturing PAGE. The first lanes represent the results of control experiments where no dNTPs were added to the reaction mixtures.

Figure 3.

Molecular models of the incoming 8-oxo-dGTP in the active site of Polη. When the incoming 8-oxo-dGTP (cyan stick) forms the anti conformation (left panels), the side chain of arginine (displayed as a purple stick) may sterically clash with O⁸ (red ball) of the incoming 8-oxo-dGTP, which may be the reason for the poor incorporation of 8-oxo-dGTP opposite template dC (green stick) (A). Proper pairing with template dC can be achieved, however, when arginine is substituted with lysine (B) or alanine (C) because of no steric hindrance. An electrostatic interaction between the lysine residue and O⁸ of 8-oxo-dGTP may facilitate the incorporation of 8-oxo-dGTP opposite template dC (B). When the incoming 8-oxo-dGTP forms the syn conformation (right panels), there appear no steric or electrostatic interactions between the amino acids and O⁸ of 8-oxo-dGTP (A–C). All models were constructed based on the crystal structure of yeast Polη [PDB#2R8J (21)]. The active site of Polη is displayed as Gaussian surface (colored according to its electric charge).