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N ‐methyl‐ N ‐nitrosourea‐lnduced mutations in a shuttle plasmid replicated in human cells
Author(s) -
Sikpi Matthew O.,
Waters Larry C.,
Kraemer Kenneth H.,
Preston R. Julian,
Mitra Sankar
Publication year - 1990
Publication title -
molecular carcinogenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.254
H-Index - 97
eISSN - 1098-2744
pISSN - 0899-1987
DOI - 10.1002/mc.2940030108
Subject(s) - biology , shuttle vector , extrachromosomal dna , microbiology and biotechnology , plasmid , mutant , mutation frequency , mutation , dna , mutagenesis , transfection , recombinant dna , cell culture , genetics , gene , vector (molecular biology)
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 O 6 ‐methylguanine‐DNA methyltransferase‐deficient cells (from 0.54 × 10 −3 to5.8 × 10 −3 ) and an 80‐fold increase when replicated in cells containing normal levels of the enzyme (from 0.047 × 10 −3 to3.8 × 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 O 6 ‐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 O 6 ‐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 O 6 ‐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.