Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Jul 15;90(14):6424–6428. doi: 10.1073/pnas.90.14.6424

An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair.

A Kat 1, W G Thilly 1, W H Fang 1, M J Longley 1, G M Li 1, P Modrich 1
PMCID: PMC46944  PMID: 8341649

Abstract

The human lymphoblastoid MT1 B-cell line was previously isolated as one of a series of mutant cells able to survive the cytotoxic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MT1 cells nevertheless remain sensitive to mutagenesis by MNNG and display a mutator phenotype. These phenotypes have been attributed to a single genetic alteration postulated to confer a defect in strand-specific mismatch repair, a proposal that attributes the cytotoxic effect of DNA alkylation in wild-type cells to futile attempts to correct mispairs that arise during replication of alkylated template strands. Our results support this view. MNNG-induced mutations in the HPRT gene of MT1 cells are almost exclusively G.C-->A.T transitions, while spontaneous mutations observed in this mutator cell line are single-nucleotide insertions, transversions, and A.T-->G.C transitions. In vitro assay has demonstrated that the MT1 line is in fact deficient in strand-specific correction of all eight base-base mispairs. This defect, which is manifest at or prior to the excision stage of the reaction, is due to simple deficiency of a required activity because MT1 nuclear extracts can be complemented by a partially purified HeLa fraction to restore in vitro repair. These findings substantiate the idea that strand-specific mismatch repair contributes to alkylation-induced cytotoxicity and imply that this process serves as a barrier to spontaneous transition, transversion, and insertion/deletion mutations in mammalian cells.

Full text

PDF
6425

Images in this article

6425Fig. 1
on p.6425
6426Fig. 2
on p.6426

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cariello N. F., Keohavong P., Kat A. G., Thilly W. G. Molecular analysis of complex human cell populations: mutational spectra of MNNG and ICR-191. Mutat Res. 1990 Aug;231(2):165–176. doi: 10.1016/0027-5107(90)90023-w. [DOI] [PubMed] [Google Scholar]
  2. Eadie J. S., Conrad M., Toorchen D., Topal M. D. Mechanism of mutagenesis by O6-methylguanine. Nature. 1984 Mar 8;308(5955):201–203. doi: 10.1038/308201a0. [DOI] [PubMed] [Google Scholar]
  3. Ellis R. E., Yuan J. Y., Horvitz H. R. Mechanisms and functions of cell death. Annu Rev Cell Biol. 1991;7:663–698. doi: 10.1146/annurev.cb.07.110191.003311. [DOI] [PubMed] [Google Scholar]
  4. Feng W. Y., Lee E. H., Hays J. B. Recombinagenic processing of UV-light photoproducts in nonreplicating phage DNA by the Escherichia coli methyl-directed mismatch repair system. Genetics. 1991 Dec;129(4):1007–1020. doi: 10.1093/genetics/129.4.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Goldmacher V. S., Cuzick R. A., Jr, Thilly W. G. Isolation and partial characterization of human cell mutants differing in sensitivity to killing and mutation by methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. J Biol Chem. 1986 Sep 25;261(27):12462–12471. [PubMed] [Google Scholar]
  6. Holmes J., Jr, Clark S., Modrich P. Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5837–5841. doi: 10.1073/pnas.87.15.5837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Karran P., Bignami M. Self-destruction and tolerance in resistance of mammalian cells to alkylation damage. Nucleic Acids Res. 1992 Jun 25;20(12):2933–2940. doi: 10.1093/nar/20.12.2933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Karran P., Marinus M. G. Mismatch correction at O6-methylguanine residues in E. coli DNA. Nature. 1982 Apr 29;296(5860):868–869. doi: 10.1038/296868a0. [DOI] [PubMed] [Google Scholar]
  9. Keohavong P., Thilly W. G. Mutational spectrometry: a general approach for hot-spot point mutations in selectable genes. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4623–4627. doi: 10.1073/pnas.89.10.4623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lahue R. S., Au K. G., Modrich P. DNA mismatch correction in a defined system. Science. 1989 Jul 14;245(4914):160–164. doi: 10.1126/science.2665076. [DOI] [PubMed] [Google Scholar]
  11. Längle-Rouault F., Maenhaut-Michel G., Radman M. GATC sequences, DNA nicks and the MutH function in Escherichia coli mismatch repair. EMBO J. 1987 Apr;6(4):1121–1127. doi: 10.1002/j.1460-2075.1987.tb04867.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Modrich P. Mechanisms and biological effects of mismatch repair. Annu Rev Genet. 1991;25:229–253. doi: 10.1146/annurev.ge.25.120191.001305. [DOI] [PubMed] [Google Scholar]
  13. Oller A. R., Thilly W. G. Mutational spectra in human B-cells. Spontaneous, oxygen and hydrogen peroxide-induced mutations at the hprt gene. J Mol Biol. 1992 Dec 5;228(3):813–826. doi: 10.1016/0022-2836(92)90866-i. [DOI] [PubMed] [Google Scholar]
  14. Plant J. E., Roberts J. J. A novel mechanism for the inhibition of DNA synthesis following methylation: the effect of N-methyl-N-nitrosourea on HeLa cells. Chem Biol Interact. 1971 Oct;3(5):337–342. doi: 10.1016/0009-2797(71)90013-5. [DOI] [PubMed] [Google Scholar]
  15. Su S. S., Lahue R. S., Au K. G., Modrich P. Mispair specificity of methyl-directed DNA mismatch correction in vitro. J Biol Chem. 1988 May 15;263(14):6829–6835. [PubMed] [Google Scholar]
  16. Thomas D. C., Roberts J. D., Kunkel T. A. Heteroduplex repair in extracts of human HeLa cells. J Biol Chem. 1991 Feb 25;266(6):3744–3751. [PubMed] [Google Scholar]
  17. Vaux D. L. Toward an understanding of the molecular mechanisms of physiological cell death. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):786–789. doi: 10.1073/pnas.90.3.786. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES