Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae

Nature Genetics volume 19, pages 384–389 (1998)Cite this article

A Correction to this article was published on 01 February 1999

Abstract

Hereditary non-polyposis colorectal cancer (HNPCC; OMIM 120435-6) is a cancer-susceptibility syndrome1 linked to inherited defects in human mismatch repair (MMR) genes2. Germline missense human MLH1 (hMLH1) mutations are frequently detected in HNPCC (ref. 3), making functional characterization of mutations in hMLH1 critical to the development of genetic testing for HNPCC. Here, we describe a new method for detecting mutations in hMLH1 using a dominant mutator effect of hMLH1 cDNA expressed in Saccharomyces cerevisiae. The majority of hMLH1 missense mutations identified in HNPCC patients abolish the dominant mutator effect. Furthermore, PCR amplification of hMLH1 cDNA from mRNA from a HNPCC patient, followed by in vivo recombination into a gap expression vector, allowed detection of a heterozygous loss-of-function missense mutation in hMLH1 using this method. This functional assay offers a simple method for detecting and evaluating pathogenic mutations in hMLH1.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: hMLH1 expression vectors and reporter vectors.
Figure 2: Mutation rate analysis.
Figure 3: Qualitative analysis of loss-of-dominant mutator effects of the hMLH1 mutations.
Figure 4: Detection of hMLH1 protein expressed in MMR-proficient YPH499 strain by immunoblot analysis with an anti-hMLH1 monoclonal antibody.
Figure 5: Schematic diagram of the method for detecting hMLH1 mutations.

Similar content being viewed by others

References

  1. Lynch, H.T., Smyrk, T. & Lynch, J.F. Overview of natural history, pathology, molecular genetics and management of HNPCC (Lynch Syndrome). Int. J. Cancer 69, 38–43 (1996).

    Article  CAS  PubMed  Google Scholar 

  2. Liu, B. et al. Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients . Nature Med. 2, 169–174 (1996).

    Article  CAS  PubMed  Google Scholar 

  3. Peltomaki, P. et al. Mutations predisposing to hereditary nonpoplyposis colorectal cancer: Database and results of a collaborative study. Gastroenterology 113, 1146–1158 (1997).

    Article  CAS  PubMed  Google Scholar 

  4. Papadopoulos, N. et al. Mutation of a mutL homolog in hereditary colon cancer. Science 263, 1625–1629 ( 1994).

    Article  CAS  PubMed  Google Scholar 

  5. Liu, B. et al. hMSH2 mutations in hereditary nonpolyposis colorectal cancer kindreds. Cancer Res. 54, 4590–4594 (1994).

    CAS  PubMed  Google Scholar 

  6. Ishioka, C. et al. Detection of heterozygous truncating mutations in the BRCA1 and APC genes by using a rapid screening assay in yeast. Proc. Natl Acad. Sci. USA 94, 2449–2453 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kolodner, R. Biochemistry and genetics of eukaryotic mismatch repair. Genes Dev. 10, 1433–1442 (1996).

    Article  CAS  PubMed  Google Scholar 

  8. Henderson, S.T. & Petes, T.D. Instability of a plasmid-borne inverted repeat in Saccharomyces cerevisiae. Genetics 134, 57–62 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Strand, M., Prolla, T.A., Liskay, R.M. & Petes, T.D. Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature 365, 274– 276 (1993).

    Article  CAS  PubMed  Google Scholar 

  10. Sia, E.A., Kokoska, R.J., Dominska, M., Greenwell, P. & Petes, T.D. Microsatellite Instability in yeast: dependence on repeat unit size and DNA mismatch repair genes. Mol. Cell. Biol. 17, 2851–2858 ( 1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ishioka, C. et al. Screening patients for heterozygous p53 mutations using a functional assay in yeast. Nature Genet. 5, 124– 129 (1993).

    Article  CAS  PubMed  Google Scholar 

  12. Pensotti, V. et al. Mean age of tumor onset in hereditary nonpolyposis colorectal cancer (HNPCC) families correlates with the presence of mutations in DNA mismatch repair genes. Genes Chromosomes Cancer 19, 135– 142 (1997).

    Article  CAS  PubMed  Google Scholar 

  13. Marsischky, G.T., Filosi, N., Kane, M.F. & Kolodner, R. Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair. Genes Dev. 10, 407– 420 (1996).

    Article  CAS  PubMed  Google Scholar 

  14. Reenan, R.A. & Kolodner, R.D. Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate mitochondrial and nuclear functions. Genetics 132, 975–985 ( 1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Lea, D.E. & Coulson, C.A. The distribution of the number of mutants in bacterial populations. J. Genet. 49, 264–285 (1949).

    Article  CAS  PubMed  Google Scholar 

  16. Christianson, T.W., Sikorski, R.S., Dante, M., Shero, J.H. & Hieter, P. Multifunctional yeast high-copy-number shuttle vectors. Gene 110, 119– 122 (1992).

    Article  CAS  PubMed  Google Scholar 

  17. Sarkar, G. & Sommer, S.S. The "megaprimer" method of site-directed mutagenesis. Biotechniques 8, 404– 407 (1990).

    CAS  PubMed  Google Scholar 

  18. Bronner, C.E. et al. Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature 368, 258–261 (1994).

    Article  CAS  PubMed  Google Scholar 

  19. Vasen, H.F. et al. The international collaborative group on hereditary non-polyposis colorectal cancer (ICG-HNPCC). Dis. Colon Rectum 34, 424–425 (1991).

    Article  CAS  PubMed  Google Scholar 

  20. Viel, A. et al. Characterization of MSH2 and MLH1 mutations in Italian families with hereditary nonpolyposis colorectal cancer. Genes Chromosomes Cancer 18, 8–18 ( 1997).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to the patients who participated in this study. We thank T. D. Petes for providing the pSH31 plasmid, N. Papadopoulos and S. Mizutani for providing cDNA containing hMLH1 mutations and E. O'Neil for technical assistance. We also thank A. Yasui, T. Noda, M. Vidal, K. Umezu, T. Hunato, S. Ishii, S. Kure and A. Horii for helpful comments about this work. This work was supported in part by Grant-in Aids for Scientific Research and International Scientific Research (Joint Study) from the Ministry of Education, Science, Sports and Culture (C.I., S.H.F., R.D.K.), Grant-in Aid from the Ministry of Health and Welfare (C.I.), the Osaka Cancer Research Foundation (C.I.), the Tokyo Biochemical Research Foundation (C.I.), the Sapporo Bioscience Foundation (C.I.) and a National Institutes of Health grant, no. GM50006 (R.D.K.).

Author information

Author notes

  1. Richard D. Kolodner

    Present address: Ludwig Institute for Cancer Research, University of California San Diego School of Medicine, La Jolla, California, 92093, USA

Authors and Affiliations

  1. Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575, Japan

    Hideki Shimodaira, Hiroyuki Shibata, Takao Suzuki, Ryunosuke Kanamaru & Chikashi Ishioka

  2. Charles A. Dana Division of Human Cancer Genetics, Dana-Farber Cancer Institute, Boston, 02115, Massachusetts, USA

    Nicole Filosi & Richard D. Kolodner

  3. Division of Experimental Oncology, Istituto Nazionale Tumori, Milano, 20133, Italy

    Paolo Radice

  4. The Seattle Project, Fred Hutchinson Cancer Research Center, Seattle, 98109, Washington, USA

    Stephen H. Friend

Authors
  1. Hideki Shimodaira
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicole Filosi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hiroyuki Shibata
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Takao Suzuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paolo Radice
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ryunosuke Kanamaru
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Stephen H. Friend
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Richard D. Kolodner
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chikashi Ishioka
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Richard D. Kolodner or Chikashi Ishioka.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shimodaira, H., Filosi, N., Shibata, H. et al. Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae. Nat Genet 19, 384–389 (1998). https://doi.org/10.1038/1277

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/1277

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing