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.

  • Article
  • Published:

Identification of Exo1-Msh2 interaction motifs in DNA mismatch repair and new Msh2-binding partners

Nature Structural & Molecular Biology volume 25, pages 650–659 (2018)Cite this article

Subjects

An Author Correction to this article was published on 04 November 2019

This article has been updated

Abstract

Eukaryotic DNA mismatch repair (MMR) involves both exonuclease 1 (Exo1)-dependent and Exo1-independent pathways. We found that the unstructured C-terminal domain of Saccharomyces cerevisiae Exo1 contains two MutS homolog 2 (Msh2)-interacting peptide (SHIP) boxes downstream from the MutL homolog 1 (Mlh1)-interacting peptide (MIP) box. These three sites were redundant in Exo1-dependent MMR in vivo and could be replaced by a fusion protein between an N-terminal fragment of Exo1 and Msh6. The SHIP-Msh2 interactions were eliminated by the msh2M470I mutation, and wild-type but not mutant SHIP peptides eliminated Exo1-dependent MMR in vitro. We identified two S. cerevisiae SHIP-box-containing proteins and three candidate human SHIP-box-containing proteins. One of these, Fun30, had a small role in Exo1-dependent MMR in vivo. The Remodeling of the Structure of Chromatin (Rsc) complex also functioned in both Exo1-dependent and Exo1-independent MMR in vivo. Our results identified two modes of Exo1 recruitment and a peptide module that mediates interactions between Msh2 and other proteins, and they support a model in which Exo1 functions in MMR by being tethered to the Msh2–Msh6 complex.

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

Fig. 1: Two regions in the S. cerevisiae Exo1 C-terminal tail mediate Msh2 interaction.
Fig. 2: SHIP box peptides inhibit Exo1-dependent MMR and mispair-promoted excision in vitro.
Fig. 3: An Exo1-Msh6 fusion can bypass the requirement for MIP and SHIP box motifs.
Fig. 4: The msh2M470I mutation disrupts the Msh2-Exo1 interaction.
Fig. 5: Bioinformatic identification of putative SHIP box peptides reveals Msh2-interacting proteins.
Fig. 6: Bioinformatic identification of putative human SHIP box peptides reveals known and potentially conserved MSH2-interacting proteins.

Similar content being viewed by others

Change history

  • 04 November 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. Goellner, E. M., Putnam, C. D. & Kolodner, R. D. Exonuclease-1-dependent and independent mismatch repair. DNA Repair 32, 24–32 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Fishel, R. Mismatch repair. J. Biol. Chem. 290, 26395–26403 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Li, Z., Pearlman, A. H. & Hsieh, P. DNA mismatch repair and the DNA damage response. DNA Repair 38, 94–101 (2016).

    PubMed  Google Scholar 

  4. Kolodner, R. D. & Marsischky, G. T. Eukaryotic DNA mismatch repair. Curr. Opin. Genet. Dev. 9, 89–96 (1999).

    CAS  PubMed  Google Scholar 

  5. Li, G. M. Mechanisms and functions of DNA mismatch repair. Cell Res. 18, 85–98 (2008).

    CAS  PubMed  Google Scholar 

  6. Spies, M. & Fishel, R. Mismatch repair during homologous and homeologous recombination. Cold Spring Harb. Perspect. Biol. 7, a022657 (2015).

    PubMed  PubMed Central  Google Scholar 

  7. Lynch, H. T., Snyder, C. L., Shaw, T. G., Heinen, C. D. & Hitchins, M. P. Milestones of Lynch syndrome: 1895–2015. Nat. Rev. Cancer 15, 181–194 (2015).

    CAS  PubMed  Google Scholar 

  8. Durno, C. A. et al. Phenotypic and genotypic characterization of biallelic mismatch repair deficiency (BMMR-D) syndrome. Eur. J. Cancer 51, 977–983 (2015).

    CAS  PubMed  Google Scholar 

  9. Waterfall, J. J. & Meltzer, P. S. Avalanching mutations in biallelic mismatch repair deficiency syndrome. Nat. Genet. 47, 194–196 (2015).

    CAS  PubMed  Google Scholar 

  10. Kane, M. F. et al. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch-repair-defective human tumor cell lines. Cancer Res. 57, 808–811 (1997).

    CAS  PubMed  Google Scholar 

  11. Orans, J. et al. Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family. Cell 145, 212–223 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Tishkoff, D. X. et al. Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2. Proc. Natl Acad. Sci. USA 94, 7487–7492 (1997).

    CAS  PubMed  Google Scholar 

  13. Genschel, J. & Modrich, P. Mechanism of 5′-directed excision in human mismatch repair. Mol. Cell 12, 1077–1086 (2003).

    CAS  PubMed  Google Scholar 

  14. Bowen, N. et al. Reconstitution of long- and short-patch mismatch repair reactions using Saccharomyces cerevisiae proteins. Proc. Natl Acad. Sci. USA 110, 18472–18477 (2013).

    CAS  PubMed  Google Scholar 

  15. Bowen, N. & Kolodner, R. D. Reconstitution of Saccharomyces cerevisiae DNA polymerase ε–dependent mismatch repair with purified proteins. Proc. Natl Acad. Sci. USA 114, 3607–3612 (2017).

    CAS  PubMed  Google Scholar 

  16. Zhang, Y. et al. Reconstitution of 5′-directed human mismatch repair in a purified system. Cell 122, 693–705 (2005).

    CAS  PubMed  Google Scholar 

  17. Constantin, N., Dzantiev, L., Kadyrov, F. A. & Modrich, P. Human mismatch repair: reconstitution of a nick-directed bidirectional reaction. J. Biol. Chem. 280, 39752–39761 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Smith, C. E. et al. Activation of Saccharomyces cerevisiae Mlh1–Pms1 endonuclease in a reconstituted mismatch repair system. J. Biol. Chem. 290, 21580–21590 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Kadyrov, F. A., Dzantiev, L., Constantin, N. & Modrich, P. Endonucleolytic function of MutLα in human mismatch repair. Cell 126, 297–308 (2006).

    CAS  PubMed  Google Scholar 

  20. Amin, N. S., Nguyen, M. N., Oh, S. & Kolodner, R. D. Exo1-dependent mutator mutations: model system for studying functional interactions in mismatch repair. Mol. Cell. Biol. 21, 5142–5155 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Wei, K. et al. Inactivation of exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility. Genes Dev. 17, 603–614 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Shell, S. S., Putnam, C. D. & Kolodner, R. D. The N terminus of Saccharomyces cerevisiae Msh6 is an unstructured tether to PCNA. Mol. Cell 26, 565–578 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Smith, C. E. et al. Dominant mutations in S. cerevisiae PMS1 identify the Mlh1–Pms1 endonuclease active site and an exonuclease-1-independent mismatch repair pathway. PLoS Genet. 9, e1003869 (2013).

    PubMed  PubMed Central  Google Scholar 

  24. Goellner, E. M. et al. PCNA and Msh2–Msh6 activate an Mlh1–Pms1 endonuclease pathway required for Exo1-independent mismatch repair. Mol. Cell 55, 291–304 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Hombauer, H., Campbell, C. S., Smith, C. E., Desai, A. & Kolodner, R. D. Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates. Cell 147, 1040–1053 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Kadyrov, F. A. et al. A possible mechanism for exonuclease-1-independent eukaryotic mismatch repair. Proc. Natl Acad. Sci. USA 106, 8495–8500 (2009).

    CAS  PubMed  Google Scholar 

  27. Schmutte, C., Sadoff, M. M., Shim, K. S., Acharya, S. & Fishel, R. The interaction of DNA mismatch repair proteins with human exonuclease 1. J. Biol. Chem. 276, 33011–33018 (2001).

    CAS  PubMed  Google Scholar 

  28. Dherin, C. et al. Characterization of a highly conserved binding site of Mlh1 required for exonuclease-1-dependent mismatch repair. Mol. Cell. Biol. 29, 907–918 (2009).

    CAS  PubMed  Google Scholar 

  29. Gueneau, E. et al. Structure of the MutLα C-terminal domain reveals how Mlh1 contributes to Pms1 endonuclease site. Nat. Struct. Mol. Biol. 20, 461–468 (2013).

    CAS  PubMed  Google Scholar 

  30. Tran, P. T. et al. A mutation in EXO1 defines separable roles in DNA mismatch repair and post-replication repair. DNA Repair 6, 1572–1583 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Gellon, L., Werner, M. & Boiteux, S. Ntg2p, a Saccharomyces cerevisiae DNA N-glycosylase and apurinic or apyrimidinic lyase involved in base-excision repair of oxidative DNA damage, interacts with the DNA mismatch repair protein Mlh1p. Identification of a Mlh1p-binding motif. J. Biol. Chem. 277, 29963–29972 (2002).

    CAS  PubMed  Google Scholar 

  32. Umar, A. et al. Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis. Cell 87, 65–73 (1996).

    CAS  PubMed  Google Scholar 

  33. Warren, J. J. et al. Structure of the human MutSα DNA lesion recognition complex. Mol. Cell 26, 579–592 (2007).

    CAS  PubMed  Google Scholar 

  34. Stormo, G. D., Schneider, T. D., Gold, L. & Ehrenfeucht, A. Use of the ‘Perceptron’ algorithm to distinguish translational initiation sites in E. coli. Nucleic Acids Res 10, 2997–3011 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).

    CAS  PubMed  Google Scholar 

  36. Kumar, A. et al. Subcellular localization of the yeast proteome. Genes Dev. 16, 707–719 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Natter, K. et al. The spatial organization of lipid synthesis in the yeast Saccharomyces cerevisiae derived from large-scale green fluorescent protein tagging and high-resolution microscopy. Mol. Cell. Proteom. 4, 662–672 (2005).

    CAS  Google Scholar 

  38. Gauci, S., Veenhoff, L. M., Heck, A. J. & Krijgsveld, J. Orthogonal separation techniques for the characterization of the yeast nuclear proteome. J. Proteome Res. 8, 3451–3463 (2009).

    CAS  PubMed  Google Scholar 

  39. Campbell, C. S. et al. Mlh2 is an accessory factor for DNA mismatch repair in Saccharomyces cerevisiae. PLoS Genet. 10, e1004327 (2014).

    PubMed  PubMed Central  Google Scholar 

  40. Dosztányi, Z., Csizmok, V., Tompa, P. & Simon, I. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 21, 3433–3434 (2005).

    PubMed  Google Scholar 

  41. Awad, S., Ryan, D., Prochasson, P., Owen-Hughes, T. & Hassan, A. H. The Snf2 homolog Fun30 acts as a homodimeric ATP-dependent chromatin-remodeling enzyme. J. Biol. Chem. 285, 9477–9484 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Chen, X. et al. The Fun30 nucleosome remodeler promotes resection of DNA double-strand break ends. Nature 489, 576–580 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Costelloe, T. et al. The yeast Fun30 and human SMARCAD1 chromatin remodelers promote DNA end resection. Nature 489, 581–584 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Araki, H. et al. Cloning DPB3, the gene encoding the third subunit of DNA polymerase II of Saccharomyces cerevisiae. Nucleic Acids Res. 19, 4867–4872 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Datta, A., Adjiri, A., New, L., Crouse, G. F. & Jinks Robertson, S. Mitotic cross-overs between diverged sequences are regulated by mismatch repair proteins in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 1085–1093 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Myung, K., Datta, A., Chen, C. & Kolodner, R. D. SGS1, the Saccharomyces cerevisiae homolog of BLM and WRN, suppresses genome instability and homeologous recombination. Nat. Genet. 27, 113–116 (2001).

    CAS  PubMed  Google Scholar 

  47. Bermudez, V. P., Farina, A., Tappin, I. & Hurwitz, J. Influence of the human cohesion establishment factor Ctf4, AND-1, on DNA replication. J. Biol. Chem. 285, 9493–9505 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Gambus, A. et al. A key role for Ctf4 in coupling the MCM2–7 helicase to DNA polymerase-α within the eukaryotic replisome. EMBO J. 28, 2992–3004 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Im, J. S. et al. Assembly of the Cdc45–Mcm2–7–GINS complex in human cells requires the Ctf4 (AND-1), RECQL4 and MCM10 proteins. Proc. Natl. Acad. Sci. USA 106, 15628–15632 (2009).

    CAS  PubMed  Google Scholar 

  50. Zhu, W. et al. Mcm10 and AND-1/CTF4 recruit DNA polymerase-α to chromatin for initiation of DNA replication. Genes Dev. 21, 2288–2299 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Chen, Z. et al. Proteomic analysis reveals a novel mutator S (MutS) partner involved in mismatch repair pathway. Mol. Cell. Proteom. 15, 1299–1308 (2016).

    CAS  Google Scholar 

  52. Traver, S. et al. MCM9 is required for mammalian DNA mismatch repair. Mol. Cell 59, 831–839 (2015).

    CAS  PubMed  Google Scholar 

  53. Jeon, Y. et al. Dynamic control of strand excision during human DNA mismatch repair. Proc. Natl Acad. Sci. USA 113, 3281–3286 (2016).

    CAS  PubMed  Google Scholar 

  54. Myler, L. R. et al. Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA-binding proteins. Proc. Natl Acad. Sci. USA 113, E1170–E1179 (2016).

    CAS  PubMed  Google Scholar 

  55. Li, G. M. & Modrich, P. Restoration of mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs. Proc. Natl Acad. Sci. USA 92, 1950–1954 (1995).

    CAS  PubMed  Google Scholar 

  56. Geng, H. et al. In vitro studies of DNA mismatch repair proteins. Anal. Biochem. 413, 179–184 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Verreault, A. De novo nucleosome assembly: new pieces in an old puzzle. Genes Dev. 14, 1430–1438 (2000).

    CAS  PubMed  Google Scholar 

  58. Hombauer, H., Srivatsan, A., Putnam, C. D. & Kolodner, R. D. Mismatch repair, but not heteroduplex rejection, is temporally coupled to DNA replication. Science 334, 1713–1716 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Javaid, S. et al. Nucleosome remodeling by hMSH2–hMSH6. Mol. Cell 36, 1086–1094 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Obmolova, G., Ban, C., Hsieh, P. & Yang, W. Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA. Nature 407, 703–710 (2000).

    CAS  PubMed  Google Scholar 

  61. Sikorski, R. S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Yan, D. & Jin, Y. Regulation of DLK-1 kinase activity by calcium-mediated dissociation from an inhibitory isoform. Neuron 76, 534–548 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Hargreaves, V. V., Shell, S. S., Mazur, D. J., Hess, M. T. & Kolodner, R. D. Interaction between the Msh2 and Msh6 nucleotide-binding sites in the Saccharomyces cerevisiae Msh2–Msh6 complex. J. Biol. Chem. 285, 9301–9310 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Antony, E. & Hingorani, M. M. Mismatch-recognition-coupled stabilization of Msh2–Msh6 in an ATP-bound state at the initiation of DNA repair. Biochemistry 42, 7682–7693 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Fien, K. & Stillman, B. Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex. Mol. Cell. Biol. 12, 155–163 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Fortune, J. M., Stith, C. M., Kissling, G. E., Burgers, P. M. & Kunkel, T. A. RPA and PCNA suppress formation of large deletion errors by yeast DNA polymerase-δ. Nucleic Acids Res 34, 4335–4341 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Gomes, X. V., Gary, S. L. & Burgers, P. M. Overproduction in Escherichia coli and characterization of yeast replication factor C lacking the ligase homology domain. J. Biol. Chem. 275, 14541–14549 (2000).

    CAS  PubMed  Google Scholar 

  68. Nakagawa, T., Flores-Rozas, H. & Kolodner, R. D. The MER3 helicase involved in meiotic crossing over is stimulated by single-stranded DNA-binding proteins and unwinds DNA in the 3′ to 5′ direction. J. Biol. Chem. 276, 31487–31493 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Jenuth, J. P. The NCBI. Publicly available tools and resources on the Web. Methods Mol. Biol. 132, 301–312 (2000).

    CAS  PubMed  Google Scholar 

  70. Cock, P. J. et al. Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 25, 1422–1423 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Katoh, K. & Standley, D. M. MAFFT: iterative refinement and additional methods. Methods Mol. Biol. 1079, 131–146 (2014).

    PubMed  Google Scholar 

  72. Scannell, D. R., Butler, G. & Wolfe, K. H. Yeast genome evolution—the origin of the species. Yeast 24, 929–942 (2007).

    CAS  PubMed  Google Scholar 

  73. Retief, J. D. Phylogenetic analysis using PHYLIP. Methods Mol. Biol. 132, 243–258 (2000).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank N. Bowen for helpful discussions and for providing many of the different protein preparations used in the in vitro MMR assays. This work was supported by NIH grants K99 ES026653 (E.M.G.), F32 CA210407 (W.J.G.) and R01 GM50006 (R.D.K.) and by the Ludwig Institute for Cancer Research (R.D.K. and C.D.P.).

Author information

Author notes

  1. Eva M. Goellner

    Present address: Toxicology and Cancer Biology, Markey Cancer Center, University of Kentucky, Lexington, KY, USA

Authors and Affiliations

  1. Ludwig Institute for Cancer Research San Diego, University of California School of Medicine, San Diego, La Jolla, CA, USA

    Eva M. Goellner, Christopher D. Putnam, William J. Graham V, Christine M. Rahal, Bin-Zhong Li & Richard D. Kolodner

  2. Department of Medicine, University of California School of Medicine, San Diego, La Jolla, CA, USA

    Christopher D. Putnam

  3. Department of Cellular and Molecular Medicine, University of California School of Medicine, San Diego, La Jolla, CA, USA

    Richard D. Kolodner

  4. Moores–UCSD Cancer Center, University of California School of Medicine, San Diego, La Jolla, CA, USA

    Richard D. Kolodner

  5. Institute of Genomic Medicine, University of California School of Medicine, San Diego, La Jolla, CA, USA

    Richard D. Kolodner

Authors
  1. Eva M. Goellner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christopher D. Putnam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William J. Graham V
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christine M. Rahal
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bin-Zhong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Richard D. Kolodner
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.M.G., C.D.P. and R.D.K. conceived the overall experimental design; E.M.G. performed strain and plasmid construction, quantitative rate measurements and two-hybrid interaction analysis; C.M.R. aided in plasmid construction and performed Rad27 and Exo1 synthetic lethality experiments; W.J.G. performed MMR assays; B.-Z.L. performed Mlh1–Pms1 focus assays; C.D.P. analyzed SHIP box sequences and evolutionary relationships; E.M.G., C.D.P. and R.D.K. wrote the paper; and all of the authors revised and modified the paper.

Corresponding author

Correspondence to Richard D. Kolodner.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Yeast two-hybrid analysis of Exo1 C-terminal deletion mutants reveals two redundant sites are involved in the Msh2-Exo1 interaction.

a, Prey vectors bearing alleles of EXO1 were co-transformed with bait vectors bearing wild-type MSH2 to test for their ability to support the Msh2-Exo1 interaction. Vectors with full-length EXO1, the exo1F447A,F448A MIP-box-defective allele and the deletion constructs eliminating residues of Exo1 C terminal to amino acid 587 interacted with Msh2, as demonstrated by growth on CSM –Leu–Trp–His medium. Note that CSM –Leu–Trp medium is a growth control. In contrast, deletion mutants affecting amino acids N terminal to amino acid 587 disrupted Msh2 binding, as did the exo1Δ571–635,Δ671–702 allele. b, Prey vectors bearing EXO1 alleles were co-transformed with bait vectors bearing wild-type MLH1 to test for their ability to support the Mlh1-Exo1 interaction. All of the tested alleles of EXO1, except for the exo1F447A,F448A MIP-box-defective allele, encoded Exo1 variants that could bind to Mlh1, as demonstrated by growth on CSM –Leu–Trp–His medium.

Supplementary Figure 2 Patch test reveals that inactivation of both the MIP and SHIP box motifs in Exo1 cause a defect in Exo1-dependent MMR but not in the ability of Exo1 to suppress the synthetic lethality of exo1Δ and rad27Δ mutations.

a, Low-copy-number ARS-CEN plasmids without an insert or bearing different EXO1 constructs were tested for their ability to suppress the mutator phenotype of the exo1Δ pol30K217E double mutant (RDKY8077) or the exo1Δ pms1A99V double mutant (RDKY4192). Empty vector and vector bearing the nuclease-dead exo1D173A allele were unable to suppress the mutator phenotype, while the wild-type EXO1 substantially suppressed the increased mutation rate of the double mutant. An allele of EXO1 containing mutations that disrupted the MIP box (exo1F447A,F448A) and deleted the region including both SHIP boxes (exo1Δ571–702) was unable to suppress the mutator phenotype, equivalent to the empty vector or the nuclease-dead exo1D173A allele. All experiments were independently repeated a minimum of two times. b, An S. cerevisiae rad27Δ exo1Δ double-mutant strain containing a wild-type EXO1-bearing URA3 plasmid was transformed with TRP1 plasmids either without EXO1 or with various EXO1 mutations. The transformed strains were then plated either on YPD or CSM medium containing 5FOA to select for cells that lost the complementing EXO1-bearing URA3 plasmid. Neither the empty vector nor the nuclease-dead exo1D173A allele could suppress the synthetic lethality of the rad27Δ exo1Δ▯double mutations and grow on 5FOA-containing medium. In contrast, alleles of EXO1 containing mutations that disrupted the MIP box (exo1F447A,F448A) or deleted the region including both SHIP boxes (exo1Δ571–702) or lacked both functional MIP or SHIP boxes (exo1F447A,F448A,Δ571–702) would support growth on 5FOA-containing medium. Thus, defects in the MIP and SHIP boxes that cause MMR defects do not disrupt the functions of Exo1 required for survival of rad27Δ strains. All experiments were independently repeated a minimum of two times.

Supplementary Figure 3 Identification of putative SHIP box sequences in Exo1, Fun30, Dpb3, Bir1 and Utp18.

Left, 2D plot of each 7-mer peptide in the proteins plotted, with the SHIP peptide motif score generated with the PSSM along the x axis and the peptide disorder score generated using IUPRED (Bioinformatics 21, 3433–3434, 2005) along the y axis. Peptides with strong scores are labeled. Right, Diagram of the proteins with the position of the putative SHIP boxes displayed as black bars over a plot of the IUPRED long-range disorder score. Most putative SHIP boxes are present in extended unstructured regions at the N or C terminus of the proteins.

Supplementary Figure 4 Yeast two-hybrid analysis of the interaction between Msh2 and Utp18 and Bir1 and analysis of the levels of Pms1–4 × GFP foci in dpb3Δ fun30Δ single-mutant strains.

a, Prey vectors bearing either BIR1 or UTP18 were co-transformed with either empty bait vectors or bait vectors bearing wild-type MSH2, and interactions were evaluated as described in the legends to Fig. 1 and Supplementary Fig. 1. All experiments were independently repeated a minimum of four times. b, Pms1–4 × GFP foci were monitored in logarithmically growing asynchronous cultures by fluorescence microscopy, and the fraction of cells with one or more Pms1–4 × GFP foci was expressed as the fold change over wild type. The average value ( ± s.d.) from three independent experiments is presented.

Supplementary Figure 5 Conservation of SHIP and MIP box sequences in proteins from the Saccharomycetacae fungi.

The conservation of SHIP and MIP boxes present in S. cerevisiae was analyzed in key Saccharomycetacae fungi, including species that diverged before the whole-genome duplication that occurred during S. cerevisiae evolution (Yeast 24, 929–942, 2007). Presence of a MIP or SHIP box motif is indicated by “Y”; absence of the motif is indicated by “N”; and the absence of a homolog is indicated by “–”. The homologs of both EXO1 and DPB3 generated by the whole-genome duplication (ohnologs), DIN7 and DLS1, respectively, are also displayed when they exist. Phylogenic relationships were derived from a previous study (G3 6, 3927–3939, 2016).

Supplementary Figure 6 Phylogenetic distribution of the SHIP boxes in MCM9 and WDHD1/CTF4 in fungi, animals and closely related eukaryotes.

A “Y” in a black box indicates that the specific clade contains the SHIP box in the homologs. An “N” in a white box indicates that the specific clade contains the homologs but lacks an identifiable SHIP box. An asterisk next to the “Y” or “N” indicates that a very small number of species have a SHIP box status that differs from that of most of the species in the clade. A dash indicates that the clade lacks homologs of these genes, such as loss of MCM8 which is an MCM9 homolog, and MCM9 genes in the Dikarya fungi.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–4

Reporting Summary

Supplementary Dataset 1

Uncropped gel images

Supplementary Dataset 2

S. cerevisiae peptide PSSM/IUPRED scores

Supplementary Dataset 3

Human peptide PSSM/IUPRED scores

Supplementary Dataset 4

Assignment of the identities of fungal Exo1 homologs

Supplementary Dataset 5

Reannotation of the gene models for some eukaryotic Exo1 homologs

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Goellner, E.M., Putnam, C.D., Graham, W.J. et al. Identification of Exo1-Msh2 interaction motifs in DNA mismatch repair and new Msh2-binding partners. Nat Struct Mol Biol 25, 650–659 (2018). https://doi.org/10.1038/s41594-018-0092-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-018-0092-y

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