Critical amino acids in human DNA polymerases eta and kappa involved in erroneous incorporation of oxidized nucleotides.
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.
Katafuchi
Atsushi
A
Division of Genetics and Mutagenesis, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan.
Sassa
Akira
A
Niimi
Naoko
N
Grúz
Petr
P
Fujimoto
Hirofumi
H
Masutani
Chikahide
C
Hanaoka
Fumio
F
Ohta
Toshihiro
T
Nohmi
Takehiko
T
D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics; D020029:Base Pairing; D020134:Catalytic Domain; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003839:Deoxyadenosines / Q000737:chemistry; D003848:Deoxyguanine Nucleotides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D000099270:Y-Family DNA Polymerases
D001120:Arginine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D003839:Deoxyadenosines; D003848:Deoxyguanine Nucleotides; C078206:8-oxodeoxyguanosine triphosphate; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases; C112338:Rad30 protein