Effects of active site amino acids and photoproduct deamination on nucleotide insertion by yeast DNA polymerase eta.

摘要

The genetic information contained in the DNA of the cell is constantly being exposed to a large variety of damaging agents: spontaneous hydrolysis, alkylating agents, UV light, oxidative species and ionizing radiations. UV light is a particular threat, due to a decrease in the ozone layer that would otherwise prevent DNA damaging UV light from reaching the earth. Organisms have developed numerous strategies to repair and synthesize past UV DNA damage.;A recently discovered member of the Y-family of DNA polymerases, polymerase eta, has been found the enzyme to be able to synthesize past cis-syn cyclobutane photodimers in DNA, unlike enzymes involved in the replication of DNA. Unlike replicative enzymes, polymerase eta has a large, open active site that appears to be able to accommodate a thymine dimer which would explain its ability to bypass this lesion. To test this hypothesis, we have mutated Ile60 in the putative dimer binding pocket to see if it can interfere with synthesis opposite a dimer, but not opposite normal DNA. We evaluated the effects of amino acid mutations I60W, I60F, and I60Y in the finger domain of the catalytic core of the yeast DNA polymerase eta on the efficiency of nucleotide insertion and synthesis past nondamaged and damaged DNA. All three mutated polymerases showed a decrease in nucleotide insertion efficiency opposite the dimer and the normal template compared to the wild-type polymerase eta. The bypass of the dimer was greatly reduced for the mutated polymerases, regardless of the template. The I60W mutant however showed a more reduced ability to insert opposite the dimer than opposite normal DNA, as expected if the tryptophan was preferentially blocking binding of the dimer.;Because polymerase eta is known to faithfully copy the bases present in a dimer, we were also interested in studying the deamination of 5-methylcytosine in a dimer, which converts a C to T and has been proposed to explain the formation of UV-induced C→T mutations. To understand better the mechanism of deamination and what influences its rate, we made use of polymerase eta to investigate the role of pH on the deamination kinetics of 5-methylcytosine-thymine containing cis-syn cyclobutane dimers in single and double stranded DNA. For both type of templates, we found the deamination rate was maximal at pH 4, in accord with a previous study of a dinucleotide, suggesting the intermediacy of a carbinolamine intermediate. We also conclude that the deamination rate was slower for double stranded DNA, due to the helical structure of the duplex DNA that hinders the nucleophilic attack.