Biological properties of imidazole ring-opened N7-methylguanine in M13mp18 phage DNA.
Guanine residues methylated at the N-7 position (7-MeGua) are susceptible to cleavage of the imidazole ring yielding 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7-MeGua). The presence of Fapy-7-MeGua in DNA template causes stops in DNA synthesis in vitro by E. coli DNA polymerase I. The biological consequences of Fapy-7-MeGua lesions for survival and mutagenesis were investigated using single-stranded M13mp18 phage DNA. Fapy-7-MeGua lesions were generated in vitro in phage DNA by dimethylsulfate (DMS) methylation and subsequent ring opening of 7-MeGua by treatment with NaOH (DMS-base). The presence of Fapy-7-MeGua residues in M13 phage DNA correlated with a significant decrease in transfection efficiency and an increase in mutation frequency in the lacZ gene, when transfected into SOS-induced JM105 E.coli cells. Sequencing analysis revealed unexpectedly, that mutation rate at guanine sites was only slightly increased, suggesting that Fapy-7-MeGua was not responsible for the overall increase in the mutagenic frequency of DMS-base treated DNA. In contrast, mutation frequency at adenine sites yielding A----G transitions was the most frequent event, 60-fold increased over DMS induced mutations. These results show that treatment with alkali of methylated single-stranded DNA generates a mutagenic adenine derivative, which mispairs with cytosine in SOS induced bacteria. The results also imply that the Fapy-7-MeGua in E. coli cells is primarily a lethal lesion.
Tudek
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B
LA147 CNRS, U140 INSERM, Institut Gustave Roussy, Villejuif, France.
Boiteux
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S
Laval
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J
D000477:Alkylating Agents / Q000494:pharmacology*; D001483:Base Sequence; D003090:Coliphages / Q000187:drug effects / Q000235:genetics*; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D007093:Imidazoles / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D011743:Pyrimidines / Q000378:metabolism; D013463:Sulfuric Acid Esters / Q000494:pharmacology*; D014162:Transfection / Q000187:drug effects; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000477:Alkylating Agents; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D007093:Imidazoles; D011743:Pyrimidines; D013463:Sulfuric Acid Esters; C071023:2,6-diamino-4-hydroxy-5-formamidopyrimidine; D006147:Guanine; C008450:7-methylguanine; C029899:imidazole; D001616:beta-Galactosidase; C007482:dimethyl sulfate