DNA damage at thymine N-3 abolishes base-pairing capacity during DNA synthesis.
3-Methylthymine was synthesized into DNA copolymers and deoxynucleoside triphosphate to study its effect on DNA synthesis by the Klenow fragment of Escherichia coli polymerase I and avian myeloblastosis virus reverse transcriptase. Both polymerases were greatly inhibited by template 3-methylthymine. In response to 3-methylthymine, misincorporation of dTTP increased slightly, but occurred only at low levels consistent with spontaneous misincorporation in vitro. Surprisingly, template 3-methylthymine resulted in a striking decrease in background misincorporation, relative to normal incorporation by the Klenow fragment, of dGTP and, to a lesser extent, of dATP and dCTP. The incorporation of 3-methyl-dTTP into DNA was studied using DNA sequencing technology. The Klenow fragment failed to incorporate 3-methyl-dTTP even at 1 mM. Reverse transcriptase incorporated 3-methyl-dTTP opposite adenine, cytosine, and thymine, but at only about 1/40,000th the efficiency of complementary deoxynucleoside triphosphate incorporation. Furthermore, synthesis generally stalled at sites of 3-methyl-thymine incorporation. From these results, we conclude that damage at the central hydrogen-bonding position of thymine abolishes its base-pairing capabilities during DNA synthesis.
D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism; D001482:Base Composition; D001483:Base Sequence; D004249:DNA Damage; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D012318:RNA Polymerase I / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012191:Retroviridae Proteins / Q000378:metabolism; D013698:Templates, Genetic; D013941:Thymine / Q000031:analogs & derivatives* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012191:Retroviridae Proteins; D012194:RNA-Directed DNA Polymerase; D012318:RNA Polymerase I; D013941:Thymine; C002347:3-methylthymine