N‐methyl‐N‐nitrosourea‐lnduced mutations in a shuttle plasmid replicated in human cells

Abstract

The supF gene of the recombinant shuttle plasmid pZ190 (modified pZ189) was used as a target to study the nature of mutations induced by N-methyl-N-nitrosourea (MNU) in human cells. Treatment of the intact plasmid with MNU followed by its replication in human lymphoblastoid cells led to extensive inactivation and no detectable mutations of the plasmid. However, exposure of the supF DNA fragment alone, followed by its ligation into the vector, caused a ten-fold increase in mutant frequency when replicated in O6-methylguanine-DNA methyltransferase-deficient cells (from 0.54 x 10(-3) to 5.8 x 10(-3)) and an 80-fold increase when replicated in cells containing normal levels of the enzyme (from 0.047 x 10(-3) to 3.8 x 10(-3)). About 45% of the mutant plasmid molecules recovered from human cells contained deletions and insertions. Sixty to 70% of the mutant molecules of wild-type size contained a single-base substitution. Most of these changes were of the G.C----A.T type, consistent with the hypothesis that O6-methylguanine is the primary mutagenic adduct induced by MNU. However, the distribution of mutation sites was highly nonrandom; more than half of all mutations were localized at the G.C position 123, and the rest were distributed in about a dozen sites. The high yield of mutations induced in the supF DNA in a host cell whose capacity for the removal of O6-methylguanine far exceeded the amount present in the supF suggests that the repair of damages in extrachromosomal DNA may be inefficient. This is supported by the observation that the yield of mutations in supF transfected into lymphoblastoid cells devoid of repair activity for O6-methylguanine was comparable to that observed with repair-proficient host cells. The present data, together with results of mutations induced in pZ189 by other agents, strongly suggest that one major determinant of mutational hot spots is the structure of the target DNA itself.

Chemical Carcinogenesis Models of Cancer: Back to the Future

“…A similar activating mutation was also seen in mammary carcinomas in rats treated with MNU (Zarbl et al 1985). These mutation patterns were in complete agreement with prior data showing that DMBA forms adducts predominantly with adenosine residues in DNA (Bigger et al 1983), whereas MNU methylates guanines, predominantly at GG dinucleotide positions (Kurowska et al 2012, Sikpi et al 1990), thus establishing a mechanistic link between carcinogen exposure and specific point mutations in cancer genes.…”

Influence of DNA repair by ada and ogt alkytransferases on the mutational specificity of alkylating agents

“…Nevertheless, our results differ markedly from those of Sikpi et al [41] and Moriwaki et al [421 in that a significant proportion of MNU-induced mutations in supFbearing plasmids propagated in human [41] and mouse [42] cells were not G:C-+A:T transitions. This difference may indicate that 06-alkylguanine lesions are more efficiently repaired in mammalian than in E. coli Celts or, al-ternatively, that lesions other than 06-alkylguanine adducts are less efficiently repaired in mammalian cells than in E. coli cells.…”