Ring-opened 7-methylguanine residues in DNA are a block to in vitro DNA synthesis.

Single-stranded M13mp18 phage DNA was methylated with dimethylsulfate (DMS), and further treated with alkali to ring-open N7-methylguanine residues and yield 2-6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) residues. Nucleotide incorporation during in vitro DNA synthesis on methylated template using E. coli DNA polymerase Klenow fragment (Kf polymerase) was reduced compared to the unmethylated template. Additional treatment of the methylated template with NaOH to generate Fapy residues, further reduced in vitro DNA synthesis compared to the synthesis on methylated templates, which suggested that Fapy residues were a block to in vitro DNA synthesis. Analysis of the termination products on sequencing gels, assuming that synthesis stops one base before a blocking lesion, indicated that arrest of DNA synthesis upon direct alkylation of single-stranded DNA occurred 1 base 3' to template adenine residues in the case of Kf polymerase and 1 base 3' to adenine and cystosine residues for T4 polymerase. When the alkylated templates were treated with NaOH to produce a template which converted all the N7-methylguanine residues to Fapy residues, the blocks to DNA synthesis were still observed one base before adenine residues. In addition to the stops previously observed for the methylated templates, however, new stops occurred one base 3' to template guanine residues for synthesis using both Kf polymerase and T4 polymerase. Fapy residues, therefore, represent a potential lethal lesion which may also arrest in vivo DNA synthesis if not repaired.     

Repair of 8-oxoG is slower in endogenous nuclear genes than in mitochondrial DNA and is without strand bias

DNA is vulnerable to the attack of certain oxygen radicals and one of the major DNA lesions formed is 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic lesion that can mispair with adenine. The repair of 8-oxoG was studied by measuring the gene specific removal of 8-oxoG after treatment of Chinese hamster ovary (CHO) fibroblasts with the photosensitizer Ro19-8022. This compound introduces 8-oxoG lesions, which can then be detected with the Escherichia coli formamidopyrimidine DNA glycosylase (FPG). In this report we present gene specific repair analysis of endogenous genes situated in different important cellular regions and also the first analysis of strand specific DNA repair of 8-oxoG in an endogenous gene. We were not able to detect any preferential repair of transcribed genes compared to non-transcribed regions and we did not detect any strand-bias in the repair of the housekeeping gene, dihydrofolate reductase (DHFR). In vivo, mitochondrial DNA is highly exposed to reactive oxygen species (ROS), and we find that the repair of 8-oxoG is more efficient in the mitochondrial DNA than in the nuclear DNA.