Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairing in vitro.
Two major stable oxidation products of 2'-deoxycytidine are 2'-deoxy-5-hydroxycytidine (5-OHdC) and 2'-deoxy-5-hydroxyuridine (5-OHdU). In order to study the in vitro incorporation of 5-OHdC and 5-OHdU into DNA by DNA polymerase, and to check the base pairing specificity of these modified bases, 5-OHdCTP and 5-OHdUTP were synthesized. Incorporation studies showed that 5-OHdCTP can replace dCTP, and to a much lesser extent dTTP, as a substrate for Escherichia coli DNA polymerase I Klenow fragment (exonuclease free). However, 5-OHdUTP can only be incorporated into DNA in place of dTTP. To study the specificity of nucleotide incorporation opposite 5-hydroxypyrimidines in template DNA, 18- and 45-member oligodeoxyribonucleotides, containing an internal 5-OHdC or 5-OHdU in two different sequence contexts, were used. Translesion synthesis past 5-OHdC and 5-OHdU in both oligonucleotides occurred, but pauses both opposite, and one nucleotide prior to, the modified base in the template were observed. The specificity of nucleotide incorporation opposite 5-OHdC and 5-OHdU in the template was sequence context dependent. In one sequence context, dG was the predominant nucleotide incorporated opposite 5-OHdC with dA incorporation also observed; in this sequence context, dA was the principal nucleotide incorporated opposite 5-OHdU. However in a second sequence context, dC was the predominant base incorporated opposite 5-OHdC. In that same sequence context, dC was also the predominant nucleotide incorporated opposite 5-OHdU. These data suggest that the 5-hydroxypyrimidines have the potential to be premutagenic lesions leading to C-->T transitions and C-->G transversions.
Purmal
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Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405-0084.
Kow
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Wallace
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D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D004247:DNA / Q000096:biosynthesis*; D004256:DNA Polymerase I / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013698:Templates, Genetic; D014498:Uracil / Q000031:analogs & derivatives* / Q000737:chemistry
D009838:Oligodeoxyribonucleotides; C045975:5-hydroxyuracil; D014498:Uracil; C017400:5-hydroxycytosine; D003596:Cytosine; D004247:DNA; D004256:DNA Polymerase I