O(6)-METHYLGUANINE IN DNA INHIBITS REPLICATION IN-VITRO BY HUMAN CELL-EXTRACTS

To study the effects of methylation damage on DNA replication in vitro , the plasmid pSVori containing the SV40 origin of replication was rea cted with N-methyl-N-nitrosourea and used as a substrate for SV40 T an tigen dependent replication by HeLa cell extracts. The plasmid was met hylated with a range of N-methyl-N-nitrosourea concentrations that int roduced an average of 0.3-2.5 O6-methylguanine and equal amounts of 3- methyladenine lesions per DNA molecule. When methylated plasmid was in cubated with extract of Mex- HeLaMR cells under conditions favoring DN A replication, an impairment of replication was observed as the accumu lation of incompletely replicated form II plasmid molecules. These ext racts simultaneously performed a T antigen independent, DpnI-sensitive DNA repair synthesis that increased with increasing DNA damage. Subtr action of this repair DNA synthesis revealed that methylation inhibite d overall replication. At low levels of methylation (less-than-or-equa l-to 1 O6-methylguanine and less-than-or-equal-to 1 3-methyladenine le sion per plasmid), inhibition was transient, while more extensive dama ge resulted in apparently irreversible inhibition of replication. Remo val of O6-methylguanine by pretreatment of the methylated plasmid with purified human O6-methylguanine-DNA methyltransferase restored replic ation to almost normal levels. When the methylated plasmid was replica ted by extracts of Mex+ HeLaS3 cells proficient in the repair of O6-me thylguanine, a lower level of inhibition and less repair DNA synthesis was observed. The inhibition of DNA synthesis and the stimulation of repair DNA synthesis are thus both largely due to the presence of O6-m ethylguanine in DNA. We suggest that the inhibition of DNA replication by O6-methylguanine may result, in part, from the processing of this lesion during DNA replication.