Multiple point mutations in a shuttle vector propagated in human cells: evidence for an error-prone DNA polymerase activity

Proc Natl Acad Sci U S A. 1987 Jul;84(14):4944-8. doi: 10.1073/pnas.84.14.4944.


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.

MeSH terms

  • Antibody Diversity
  • Cell Line
  • DNA Damage
  • DNA Repair*
  • DNA-Directed DNA Polymerase / metabolism*
  • Fibroblasts / metabolism
  • Genetic Vectors* / radiation effects
  • Humans
  • Lymphocytes / metabolism
  • Mutation*
  • Plasmids* / radiation effects
  • Ultraviolet Rays
  • Xeroderma Pigmentosum / metabolism


  • DNA-Directed DNA Polymerase

Associated data

  • GENBANK/M16969