DNA synthesis with methylated poly(dC-dG) templates. Evidence for a competitive nature to miscoding by O(6)-methylguanine.
The alternating copolymer poly(dC-dG) has been methylated with either dimethyl sulphate or N-methyl-N-nitrosourea and the levels of the various methylation products determined. In addition to the 3-methylcytosine, 3-methylguanine and 7-methylguanine (produced by both agents) reaction with N-methyl-N-nitrosourea also yielded easily detectable amounts of O(6)-methylguanine and phosphotriesters. These methylated polymers were then used as templates in an in vitro assay with Escherichia coli DNA polymerase I measuring the incorporation of complementary (dCMP and dGMP) and noncomplementary (dAMP and dTMP) nucleotides. When the dimethyl sulphate-methylated polymer was used as template there was virtually no detectable incorporation of non-complementary nucleotides indicating that no miscoding could be attributed to the presence of 3-methylcytosine, 3-methylguanine or 7-methylguanine. However, when the N-methyl-N-nitrosourea-methylated polymer was used as template there was a specific incorporation of dTMP but not of dAMP. The amount of dTMP incorporated was always less than the level of O(6)-methylguanine in the template and was found to vary with the relative concentrations of the deoxynucleoside 5'-triphosphates in the assay. As the amount of dCTP present in the assay was decreased the wrong incorporation of dTMP increased and approached the level that would have been expected for a one-to-one miscoding by O(6)-methylguanine as the concentration of dCTP approached zero. The results indicate that O(6)-methylguanine is capable of miscoding with a DNA polymerase but the miscoding is competitive with the normal incorporation of dCMP: when the 5'-triphosphate precursors are present in equal amounts approximately one O(6)-methylguanine in three miscodes leading to the incorporation of dTMP.
D004247:DNA / Q000096:biosynthesis*; D004256:DNA Polymerase I / Q000378:metabolism; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D004926:Escherichia coli / Q000201:enzymology; D005815:Genetic Code; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D008745:Methylation; D008770:Methylnitrosourea; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D013463:Sulfuric Acid Esters; D013698:Templates, Genetic; D013938:Thymidine Monophosphate / Q000378:metabolism
D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D011089:Polydeoxyribonucleotides; D013463:Sulfuric Acid Esters; C019530:poly(dC-dG); D013938:Thymidine Monophosphate; D006147:Guanine; D008770:Methylnitrosourea; D004247:DNA; C008449:O-(6)-methylguanine; D004256:DNA Polymerase I