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2005 | 52 | 3 | 575-583
Article title

MutS as a tool for mutation detection

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Abstracts
EN
MutS, a DNA mismatch-binding protein, seems to be a promising tool for mutation detection. We present three MutS based approaches to the detection of point mutations: DNA retardation, protection of mismatched DNA against exonuclease digestion, and chimeric MutS proteins. DNA retardation in polyacrylamide gels stained with SYBR-Gold allows mutation detection using 1-3 µg of Thermus thermophilus his6-MutS protein and 50-200 ng of a PCR product. The method enables the search for a broad range of mutations: from single up to several nucleotide, as mutations over three nucleotides could be detected in electrophoresis without MutS, due to the mobility shift caused by large insertion/deletion loops in heteroduplex DNA. The binding of DNA mismatches by MutS protects the complexed DNA against exonuclease digestion. The direct addition of the fluorescent dye, SYBR-Gold, allows mutation detection in a single-tube assay. The limited efficiency of T4 DNA polymerase as an exonuclease hampers the application of the method in practice. The assay required 300-400 ng of PCR products in the range of 200-700 bp and 1-3 µg of MutS. MutS binding to mismatched DNA immobilised on a solid phase can be observed thanks to the activity of a reporter domain linked to MutS. We obtained chimeric bifunctional proteins consisting of T. thermophilus MutS and reporter domains, like β-galactosidase or GFP. Very low detection limits for β-galactosidase could theoretically enable mutation detection not only by the examination of PCR products, but even of genomic DNA.
Keywords
Year
Volume
52
Issue
3
Pages
575-583
Physical description
Dates
published
2005
received
2005-03-15
revised
2005-07-14
accepted
2005-07-22
References
  • Allen DJ, Makhov A, Grilley M, Taylor J, Thresher R, Modrich P, Griffith JD (1997) MutS mediates heteroduplex loop formation by a translocation mechanism. EMBO J 16: 4467-4476.
  • Au KG, Welsh K, Modrich P (1992) Initiation of methyl-di-rected mismatch repair. J Biol Chem 267: 12142-12148.
  • Behrensdorf HA, Pignot M, Windhab N, Kappel A (2000) Rapid parallel mutation scanning of gene fragments using a microelectronic protein-DNA chip format. Nucleic Acids Res 30: E64.
  • Bi LJ, Zhou YF, Zhang XE, Deng JY, Zhang ZP, Xie B, Zhang CG (2003) A MutS-based protein chip for detection of DNA mutations. Anal Chem 75: 4113-4119.
  • Biswas I, Hsieh P (1997) Interaction of MutS protein with the major and minor grooves of a heteroduplex DNA. J Biol Chem 272: 13355-13364.
  • Biswas I, Fleming KG, Qin J, Lary JW, Yphantis DA, Yang W, Hsieh P (1999) Oligomerization of a MutS mismatch repair protein from Thermus aquaticus. J Biol Chem 274: 23673-23678. Thermus aquaticus.
  • Biswas I, Obmolova G, Takahashi M, Herr A, Newman MA, Yang W, Hsieh P (2001) Disruption of helix-u--turn-helix motif of MutS protein: loss of subunit dimerization, mismatch binding and ATP hydrolysis. J Mol Biol 305: 805-816.
  • Bjornson KP, Blackwell LJ, Sage H, Baitinger C, Allen D, Modrich P (2003) Assembly and molecular activities of the MutS tetramer. J Biol Chem 278: 34667-34673.
  • Blackwell LJ, Bjornson KP, Allen DJ, Modrich P (2001) Distinct MutS DNA-binding modes that are differentially modulated by binding and hydrolysis. J Biol Chem 276: 34339-34347.
  • Bronstein I, Martin CS, Fortin JJ, Olesen CE, Voyta JC (1996) Chemiluminescence: sensitive detection technology for reporter gene assays. Clin Chem 42: 1542-1546.
  • Brown J, Brown T, Fox KR (2001) Affinity of mismatch-binding protein MutS for heteroduplexes containing different mismatches. Biochem J 354: 627-633.
  • Buermeyer AB, Dêschenes SM, Baker SM, Liskay RM (1999) Mammalian DNA mismatch repair. Annu Rev Genet 33: 533-564.
  • Connor M, Ferguson-Smith M (1997) Essential Medical Genetics, 5th edn, Table 21.1, Blackwell Science, Oxford.
  • Corchero JL, Villaverde A (1999) Tolerance of Escherichia coli β-galactosidase C-terminus to different-sized fusions. Biotechnol Bioeng 64: 644-649.
  • Duckett DR, Drummond JT, Murchie AI, Reardon JT, San-car A, Lilley DM, Modrich P (1996) Human MutSα recognizes damaged DNA base pairs containing O6-meth-ylguanine, O4-methylthymine, or the cisplatin-d (GpG) adduct. Proc Natl Acad Sci USA 93: 6443-6447.
  • Ellis LA, Taylor GR, Banks R, Baumberg S (1994) MutS binding protects heteroduplex DNA from exonuclease digestion in vitro: a simple method for detecting mutations. Nucleic Acids Res 22: 2710-2711.
  • Feng G, Winkler ME (1995) Single step purifications of his6-MutH, his6-MutL and his6-MutS repair proteins of Escherichia coli K12. Biotechniques 19: 956-965.
  • Fries JR, Brand L, Eggeling C, Köllner M, Seidel CAM (1998) Quantitative identification of different single molecules by selective time-resolved confocal fluorescence spectroscopy. J Phys Chem A 102: 6601-6613.
  • Geschwind DH, Rhee R, Nelson SF (1996) A biotinylated MutS fusion protein and its use in a rapid mutation screening technique. Genet Anal 13: 105-111.
  • Gotoh M, Hasebe M, Ohira T, Hasegawa Y, Shinohara Y, Sota H, Nakao J, Tosu M (1997) Rapid method for detection of point mutation using mismatch binding protein (MutS) and an optical biosensor. Genet Anal 14: 47-50.
  • Gotoh K, Hata M, Miyajima M, Yokota H (2000) Genome wide detection of unknown subtle mutations in bacteria by combination of MutS and RDA. Biochem Biophys Res Commun 268: 535-540.
  • Iwane AH, Funatsu T, Harada Y, Tokunaga M, Ohara O, Morimoto S, Yanagida T (1997) Single molecular assay of individual ATP turnover by a myosin-GFP fusion protein expressed in vitro. FEBS Lett 407: 235-238.
  • Jain VK, Magrath, IT (1991) A chemiluminescent assay for quantitation of β-galactosidase in the femtogram range: application to quantitation of β-galactosidase in lacZtransfected cells. Anal Biochem 199: 119-124.
  • Johnson KA, Mierzwa ML, Fink SP, Marnett LJ, Hancock AB Jr (1999) MutS recognition of exocyclic DNA adducts that are endogenous products of lipid oxidation. J Biol Chem 274: 27112-27118.
  • Joshi A, Rao BJ (2001) MutS recognition: multiple mismatches and sequence context effects. J Biosci 26: 595--606.
  • Kemper B (1997) In DNA Damage and Repair. Biochemistry, Genetics and Cell Biology. Nickoloff JA, Hoekstra M, eds, pp 179-204. Humana Press, Totowa, NJ.
  • Kubitscheck U, Kückmann O, Kues T, Peters R (2000) Imaging and Tracking of Single GFP Molecules in Solution. Biophys J 78: 2170-2179.
  • Lamers MH, Perrakis A, Enzlin JH, Winterwerp HH, de Wind N, Sixma TK (2000) The crystal structure of mismatch repair protein MutS binding to a GxT mismatch. Nature 407: 711-717.
  • Lishanski A, Ostrander EA, Rine J (1994) Mutation detection by mismatch binding protein, MutS, in amplified DNA: application to the cystic fibrosis gene. Proc Natl Acad Sci USA 91: 2674-2678.
  • McKusick VA (1994). Mendelian Inheritance in Man: A Catalog of Human Genetics and Genetic Disorders, 11th edn. The Johns Hopkins University Press, Baltimore.
  • Modrich, P (1991) Mechanisms and biological effects of mismatch repair. Annu Rev Genet 25: 229-253.
  • Nakahara T, Zhang, QM, Hashiguchi K, Yonei S (2000) Identification of proteins of Escherichia coli and Sacharomyces cerevisiae that specifically bind to C/C mismatches in DNA. Nucleic Acids Res 28: 2551-2556.
  • Obmolova G, Ban C, Hsieh P, Yang W (2000) Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA. Nature 407: 703-710.
  • Parker BO, Marinus MG (1992) Repair of DNA heteroduplexes containing small heterologous sequences in Escherichia coli. Proc Natl Acad Sci USA 89: 1730-1734.
  • Parsons BL, Heflich RH (1997) Evaluation of MutS as a tool for direct measurement of point mutations in genomic DNA. Mutat Res 374: 277-285.
  • Parsons BL, Heflich RH (1998) Detection of basepair substitution mutation at a frequency of 1 x 10-7by combining two genotypic selection methods, MutEx enrichment and allele-specific competitive blocker PCR. Environ Mol Mutagen 32: 200-211
  • Roses AD (2001) Pharmacogenetics. Hum Mol Genet 10: 2261-2267.
  • Sachadyn P, Stanisławska A, Kur J (2000) One tube mutation detection using sensitive fluorescent dyeing of MutS protected DNA. Nucleic Acids Res 28: E36.
  • Smith J, Modrich P (1997) Removal of polymerase-pro-duced mutant sequences from PCR products. Proc Natl Acad Sci USA 94: 6847-6850.
  • Stanisławska-Sachadyn A, Sachadyn P, Jędrzejczak R, Kur J (2003) Construction and purification of his6-Thermus thermophilus MutS protein. Protein Expr Purif 28: 69-77.
  • Stanisławska-Sachadyn A, Paszko Z, Kluska A, Skasko E, Sromek M, Balabas A, Janiec-Jankowska A, Wiśniewska A, Kur J, Sachadyn P (2005) Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples. Mutat Res 570: 97-103.
  • Su S, Lahue RS, Au KG, Modrich P (1988) Mispair specificity of methyl-directed DNA mismatch correction in vitro. J Biol Chem 263: 6829-6835.
  • Sun HB, Yokota H (2000) MutS-mediated detection of DNA mismatches using atomic force microscopy. Anal Chem 72: 3138-3141.
  • Takamatsu S, Kato R, Kuramitsu S (1996) Mismatch DNA recognition protein from an extremely thermophilic bacterium, Thermus thermophilus HB8. Nucleic Acids Res 24: 640-647.
  • Viaplana E, Feliu JX, Corchero JL, Villaverde, A (1997) Reversible activation of a cryptic cleavage site within E. coli β-galactosidase in β-galactosidase fusion proteins. Biochim Biophys Acta 1343: 221-226.
  • Vijayvargia R, Biswas I (2002) MutS2 family protein from Pyrococcus furiosus. Curr Microbiol 44: 224-228.
  • Wagner R, Debbie P, Radman M (1995) Mutation detection using immobilized mismatch binding protein (MutS). Nucleic Acids Res 23: 3944-3948.
  • Wang J, Liu J (2004) Directly fishing out subtle mutations in genomic DNA with histidine-tagged Thermus thermophilus MutS. Mutat Res 547: 41-47.
  • Wabuyele MB, Ford SM, Stryjewski W, Barrow J, Soper SA (2001) Single molecule detection of double-stranded DNA in poly (methylmethacrylate) and polycarbonate microfluidic devices. Electrophoresis 22: 3939-3948.
  • Whitehouse A, Deeble J, Parmar R, Taylor GR, Markham AF, Meredith DM (1997) Analysis of the mismatch and insertion/deletion binding properties of Thermus thermophilus, HB8, MutS. Biochem Biophys Res Commun 233: 834-837.
  • Worth L Jr, Bader T, Yang J, Clark S (1998) Role of MutS ATPase activity in MutS,L-dependent block of in vitro strand transfer. J Biol Chem 273: 23176-23182..
  • Wu TH, Marinus MG (1999) Deletion mutation analysis of the mutS Gene in Escherichia coli. J Biol Chem 274: 5948-5952.
Document Type
Publication order reference
YADDA identifier
bwmeta1.element.bwnjournal-article-abpv52i3p575kz
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