To investigate the effect of DNA replication on the mutation spectrum induced in diploid human fibroblasts by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), cells were synchronized and exposed to MNNG either at the G1-S border or in late S phase, and the mutations in the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene were examined. The coding regions of 92 independent mutants were characterized by direct sequencing of mRNA-polymerase chain reaction-amplified complementary DNA. While there was little difference in the sensitivity of the two populations to the cytotoxic effects of MNNG, the frequency of mutants induced in late S populations was significantly lower than that induced in G1-S populations. The majority of induced complementary DNA mutations were single base substitutions (54%) and splice site mutations (43%). Analysis of the intron-exon boundaries of more than one-half of the splicing mutants showed that almost all contained base substitutions in the hprt gene. A broad mutational spectrum was observed in low-dose (4, 6, or 8 µm) treatments; only 27% were G to A transitions, whereas 80% of base substitutions derived from the high-dose (10 or 12 µm) treatments were G to A transitions in G1-S populations. An intermediate frequency (64%) of G to A transitions was observed in late S populations exposed to MNNG. When the causative premutation lesion was O6-methylguanine, 75% of G to A transitions that were observed in G1-S populations clustered on both the transcribed and the nontranscribed strands of the 5′ half of the hprt gene. In contrast, 50% of G to A transitions were located only on the nontranscribed strand of this region in late S populations. The results indicate that O6-alkylguanine-DNA-alkyltransferase may not efficiently remove O6-methylguanine from the 5′ half of the gene but can repair lesions far away from this region during initiation of replication. Our results are consistent with the notion that the putative origin of replication is located at intron 1 of the hprt gene.

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This research was supported in part by Grants NSC 79-0203-B001-12 and NSC 81-0211-B-007-07 from the National Science Council, Republic of China.

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