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

Author: Katafuchi Atsushi  

Publisher: Oxford University Press

ISSN: 1362-4962

Source: Nucleic Acids Research, Vol.38, Iss.3, 2010-01, pp. : 859-867

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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 Pol and Pol proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Pol 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 Pol) to 7 : 1 (R61K). Similarly, Tyr112 in Pol was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Pol) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Pol, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Pol and Pol might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.

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