Multiple point mutations in a shuttle vector propagated in human cells: evidence for an error-prone DNA polymerase activity.
Mutagenesis was studied at the DNA-sequence level in human fibroblast and lymphoid cells by use of a shuttle vector plasmid, pZ189, containing a suppressor tRNA marker gene. In a series of experiments, 62 plasmids were recovered that had two to six base substitutions in the 160-base-pair marker gene. Approximately 20-30% of the mutant plasmids that were recovered after passing ultraviolet-treated pZ189 through a repair-proficient human fibroblast line contained these multiple mutations. In contrast, passage of ultraviolet-treated pZ189 through an excision-repair-deficient (xeroderma pigmentosum) line yielded only 2% multiple base substitution mutants. Introducing a single-strand nick in otherwise unmodified pZ189 adjacent to the marker, followed by passage through the xeroderma pigmentosum cells, resulted in about 66% multiple base substitution mutants. The multiple mutations were found in a 160-base-pair region containing the marker gene but were rarely found in an adjacent 170-base-pair region. Passing ultraviolet-treated or nicked pZ189 through a repair-proficient human B-cell line also yielded multiple base substitution mutations in 20-33% of the mutant plasmids. An explanation for these multiple mutations is that they were generated by an error-prone polymerase while filling gaps. These mutations share many of the properties displayed by mutations in the immunoglobulin hypervariable regions.
D000916:Antibody Diversity; D002460:Cell Line; D004249:DNA Damage; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors / Q000528:radiation effects; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000528:radiation effects; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000378:metabolism
D004259:DNA-Directed DNA Polymerase