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.

  • Original Article
  • Published:

Flanking sequence specificity determines coding microsatellite heteroduplex and mutation rates with defective DNA mismatch repair (MMR)

Oncogene volume 29, pages 2172–2180 (2010)Cite this article

Subjects

Abstract

The activin type II receptor (ACVR2) contains two identical microsatellites in exons 3 and 10, but only the exon 10 microsatellite is frameshifted in mismatch repair (MMR)-defective colonic tumors. The reason for this selectivity is not known. We hypothesized that ACVR2 frameshifts were influenced by DNA sequences surrounding the microsatellite. We constructed plasmids in which exons 3 or 10 of ACVR2 were cloned +1 bp out of frame of enhanced green fluorescent protein (EGFP), allowing –1 bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells, and subsequent non-fluorescent cells were sorted, cultured and harvested for mutation analysis. We swapped DNA sequences flanking the exon 3 and 10 microsatellites to test our hypothesis. Native ACVR2 exon 3 and 10 microsatellites underwent heteroduplex formation (A7/T8) in hMLH1−/− cells, but only exon 10 microsatellites fully mutated (A7/T7) in both hMLH1−/− and hMSH6−/− backgrounds, showing selectivity for exon 10 frameshifts and inability of exon 3 heteroduplexes to fully mutate. Substituting nucleotides flanking the exon 3 microsatellite for nucleotides flanking the exon 10 microsatellite significantly reduced heteroduplex and full mutation in hMLH1−/− cells. When the exon 3 microsatellite was flanked by nucleotides normally surrounding the exon 10 microsatellite, fully mutant exon 3 frameshifts appeared. Mutation selectivity for ACVR2 lies partly with flanking nucleotides surrounding each microsatellite.

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
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

Abbreviations

TGFBR2:

transforming growth factor β receptor 2

ACVR2:

activin type II receptor

MMR:

mismatch repair system

MSI:

microsatellite instability

IDL:

insertion deletion loop

MR:

mutation resistant

IF:

in frame of EGFP

OF:

out of frame of EGFP

EGFP:

enhanced green fluorescent protein

CRC:

colorectal cancer

References

  • Acharya S, Wilson T, Gradia S, Kane MF, Guerrette S, Marsischky GT et al. (1996). hMSH2 forms specific mispair-binding complexes with hMSH3 and hMSH6. Proc Natl Acad Sci USA 93: 13629–13634.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Akiyama Y, Sato H, Yamada T, Nagasaki H, Tsuchiya A, Abe R et al. (1997). Germ-line mutation of the hMSH6/GTBP gene in an atypical hereditary nonpolyposis colorectal cancer kindred. Cancer Res 57: 3920–3923.

    CAS  PubMed  Google Scholar 

  • Carethers JM, Chauhan DP, Fink D, Nebel S, Bresalier RS, Howell SB et al. (1999). Mismatch repair proficiency and in vitro response to 5-fluorouracil. Gastroenterology 117: 123–131.

    Article  CAS  PubMed  Google Scholar 

  • Chung H, Young DJ, Lopez CG, Le TA, Lee JK, Ream-Robinson D et al. (2008). Mutation rates of TGFBR2 and ACVR2 coding microsatellites in human cells with defective DNA mismatch repair. PLoS ONE 3: e3463.

    Article  PubMed  PubMed Central  Google Scholar 

  • Fishel R, Lescoe MK, Rao MR, Copeland NG, Jenkins NA, Garber J et al. (1993). The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 75: 1027–1038.

    Article  CAS  PubMed  Google Scholar 

  • Grady WM, Carethers JM . (2008). Genomic and epigenetic instability in colorectal cancer pathogenesis. Gastroenterology 135: 1079–1099.

    Article  CAS  PubMed  Google Scholar 

  • Harrington JM, Kolodner RD . (2007). Saccharomyces cerevisiae Msh2-Msh3 acts in repair of base-base mispairs. Mol Cell Biol 27: 6546–6554.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hempen PM, Zhang L, Bansal RK, Iacobuzio-Donahue CA, Murphy KM, Maitra A et al. (2003). Evidence of selection for clones having genetic inactivation of the activin A type II receptor (ACVR2) gene in gastrointestinal cancers. Cancer Res 63: 994–999.

    CAS  PubMed  Google Scholar 

  • Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa JP et al. (1998). Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci USA 95: 6870–6875.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jung B, Doctolero RT, Tajima A, Nguyen AK, Keku T, Sandler RS et al. (2004). Loss of activin receptor type 2 protein expression in microsatellite unstable colon cancers. Gastroenterology 126: 654–659.

    Article  CAS  PubMed  Google Scholar 

  • Jung B, Smith EJ, Doctolero RT, Gervaz P, Alonso JC, Miyai K et al. (2006). Influence of target gene mutations on survival, stage and histology in sporadic microsatellite unstable colon cancers. Int J Cancer 118: 2509–2513.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jung BH, Beck SE, Cabral J, Chau E, Cabrera BL, Fiorino A et al. (2007). Activin type 2 receptor restoration in MSI-H colon cancer suppresses growth and enhances migration with activin. Gastroenterology 132: 633–644.

    Article  CAS  PubMed  Google Scholar 

  • Luria SE, Delbruck M . (1943). Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28: 491–511.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J et al. (1995). Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science 268: 1336–1338.

    Article  CAS  PubMed  Google Scholar 

  • Marsischky GT, Kolodner RD . (1999). Biochemical characterization of the interaction between the Saccharomyces cerevisiae MSH2-MSH6 complex and mispaired bases in DNA. J Biol Chem 274: 26668–26682.

    Article  CAS  PubMed  Google Scholar 

  • Modrich P . (2006). Mechanisms in eukaryotic mismatch repair. J Biol Chem 281: 30305–30309.

    Article  CAS  PubMed  Google Scholar 

  • Nicolaides NC, Papadopoulos N, Liu B, Wei YF, Carter KC, Ruben SM et al. (1994). Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature 371: 75–80.

    Article  CAS  PubMed  Google Scholar 

  • Palombo F, Iaccarino I, Nakajima E, Ikejima M, Shimada T, Jiricny J . (1996). hMutSbeta, a heterodimer of hMSH2 and hMSH3, binds to insertion/deletion loops in DNA. Curr Biol 6: 1181–1184.

    Article  CAS  PubMed  Google Scholar 

  • Papadopoulos N, Nicolaides NC, Wei YF, Ruben SM, Carter KC, Rosen CA et al. (1994). Mutation of a mutL homolog in hereditary colon cancer. Science 263: 1625–1629.

    Article  CAS  PubMed  Google Scholar 

  • Parsons R, Myeroff LL, Liu B, Willson JK, Markowitz SD, Kinzler KW et al. (1995). Microsatellite instability and mutations of the transforming growth factor beta type II receptor gene in colorectal cancer. Cancer Res 55: 5548–5550.

    CAS  PubMed  Google Scholar 

  • Thibodeau SN, Bren G, Schaid D . (1993). Microsatellite instability in cancer of the proximal colon. Science 260: 816–819.

    Article  CAS  PubMed  Google Scholar 

  • Veigl ML, Kasturi L, Olechnowicz J, Ma AH, Lutterbaugh JD, Periyasamy S et al. (1998). Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. Proc Natl Acad Sci USA 95: 8698–8702.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Woerner SM, Kloor M, Mueller A, Rueschoff J, Friedrichs N, Buettner R et al. (2005). Microsatellite instability of selective target genes in HNPCC-associated colon adenomas. Oncogene 24: 2525–2535.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported by the United States Public Health Service (DK067287 to JMC), the UCSD Digestive Diseases Research Development Center (DK080506), and the VA Research Service (Merit Review Award to JMC). We thank the support of Moores UCSD Cancer Center Flow Cytometry and DNA Sequencing Shared Resources.

Author contributions: Chung H and Carethers JM designed the research; Chung H, Young DJ, Lopez CG, Lai J, Holmstrom J, Cabrera BL and Ream-Robinson D performed the research; Chung H, Lopez CG and Carethers JM analyzed data; and Chung H and Carethers JM wrote the paper. All authors have approved the final version of this paper.

Author information

Authors and Affiliations

  1. Department of Medicine, University of California, San Diego, CA, USA

    H Chung, C G Lopez, J F Lai, J Holmstrom, D Ream-Robinson, B L Cabrera & J M Carethers

  2. Rebecca and John Moores Comprehensive Cancer Center, University of California, San Diego, CA, USA

    D J Young & J M Carethers

  3. VA San Diego Healthcare System, San Diego, CA, USA

    J M Carethers

Authors
  1. H Chung
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C G Lopez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D J Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J F Lai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J Holmstrom
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. D Ream-Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. B L Cabrera
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. J M Carethers
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J M Carethers.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chung, H., Lopez, C., Young, D. et al. Flanking sequence specificity determines coding microsatellite heteroduplex and mutation rates with defective DNA mismatch repair (MMR). Oncogene 29, 2172–2180 (2010). https://doi.org/10.1038/onc.2009.508

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2009.508

Keywords

This article is cited by

Search

Advanced search

Quick links