The role of pre-replication and post-replication processes in mutation induction in Haemophilus influenzae by N-methyl-N'-nitro-N-nitrosoguanidine. |
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Authors: | R F Kimball S W Perdue M E Boling |
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Affiliation: | Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 27830, U.S.A. |
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Abstract: | Studies were carried out on the repair and fixation of premutational damage induced in Haemophilus influenzae by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The studies employed a temperature-sensitive DNA elongation mutant (dna9) and its combinations with mutants defective in pyrimidine dimer excision (uvr1, uvr2) and in recombination (rec1). The dna9 mutant is shown to be leaky, allowing about 1% of the normal rate of DNA synthesis at the restrictive temperature. Repair of premutational lesions was detected by a decline in mutation frequency with increasing delay in DNA replication in dna9 at the restrictive temperature. This repair is unaffected by the pyrimidine dimer excision system. Mutation fixation was detected by the ability of DNA from treated and then lysed cells to transfer mutants to recipient cells by transformation. Some fixation occurred at the restrictive temperature but much less than at the non-restrictive temperature suggesting that an appreciable minority of the mutations resulted from lesions introduced near the replication fork but that the majority of mutations arise from lesions introduced at some distance from the fork, perhaps randomly. The DNA synthesized immediately after MNNG treatment is of lower molecular weight than normal and returns to normal with time. This return is blocked in the rec1 mutant, suggesting that recombination is involved. The possible role of this process in MNNG mutagenesis is discussed. |
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Keywords: | MNNG NC TMB trismaleate buffer NHI brain-heart infusion dThd thymidine BrdUrd 5-bromodeoxyuridine TCA trichloroacetic acid |
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