goat anti-rabbit IgG Alexa Fluor 555. Before mounting TO-PRO- 3 (Invitrogen, Germany) diluted 1:1000 in PBS was added to all coverslipes for 1 h at room temperature to counterstain nucleic acids and coverslipes were washed again with PBS. After that, samples were mounted with ProLong Gold (Invitrogen, Germany) and examined using a Leica TCS-NT confocal microscope (Leica Lasertechnik GmbH, Heidelberg, Germany). Control for subcellular localization was carried out using unlabeled MutLa and mock control for fluorescent labeled MutLa was determined with the pECFP-C1/-N1- and pDSRed- C1/-N1- vectors. MMR Assay To analyze the consequences of GFP or Red labeling different MutLa variants were tested in vitro for MMR ability as described before [23]. Briefly, 50 mg of HEK293T nuclear extract, which is deficient in mismatch repair [21] was supplemented with 5 mg whole protein extract from HEK293T cells expressing recombi- nant MutLa constructs. A substrate plasmid bearing a G-T mismatch within an EcoRV restriction site was added to these protein compositions, which is restored when repair occurs directed by a 39 single-strand nick at a distance of 83 bp to the mismatch. Reactions were incubated at 37uC for 20 min and terminated with 50 ml stop-buffer (24 mM EDTA pH 8.0, 0.7% SDS, 2 mg/ml proteinase K) by an additional incubation for 15 min at 37uC. Plasmids were extracted from the reaction mixture by phenol-chloroform extraction and purified by ethanol co-precipitation with tRNA. Subsequent digestion with EcoRV as well as AseI produced two smaller fragments besides the linearized vector (which is generated by AseI digestion) when repair was References 1. Lynch HT, de la Chapelle A (2003) Hereditary colorectal cancer. N Engl J Med 348: 919–932. 2. Mohd AB, Palama B, Nelson SE, Tomer G, Nguyen M, et al. (2006) Truncation of the C-terminus of human MLH1 blocks intracellular stabilization of PMS2 and disrupts DNA mismatch repair. DNA Repair (Amst) 5: 347–361. 3. Brieger A, Adryan B, Wolpert F, Passmann S, Zeuzem S, et al. (2010) Cytoskeletal scaffolding proteins interact with Lynch-Syndrome associated mismatch repair protein MLH1. Proteomics 10: 3343–3355. 4. Cannavo E, Gerrits B, Marra G, Schlapbach R, Jiricny J (2007) Characteriza- tion of the interactome of the human MutL homologues MLH1, PMS1, and PMS2. J Biol Chem 282: 2976–2986. 5. Mac Partlin M, Homer E, Robinson H, McCormick CJ, Crouch DH, et al. (2003) Interactions of the DNA mismatch repair proteins MLH1 and MSH2 with c-MYC and MAX. Oncogene 22: 819–825. 6. Her C, Vo AT, Wu X (2002) Evidence for a direct association of hMRE11 with the human mismatch repair protein hMLH1. DNA Repair (Amst) 1: 719–729. 7. Brieger A, Plotz G, Zeuzem S, Trojan J (2007) Thymosin beta 4 expression and nuclear transport are regulated by hMLH1. Biochem Biophys Res Commun 364: 731–736. 8. Hong Z, Jiang J, Hashiguchi K, Hoshi M, Lan L, et al. (2008) Recruitment of mismatch repair proteins to the site of DNA damage in human cells. J Cell Sci 121: 3146–3154. 9. Ou J, Rasmussen M, Westers H, Andersen SD, Jager PO, et al. (2009) Biochemical characterization of MLH3 missense mutations does not reveal an apparent role of MLH3 in Lynch syndrome. Genes Chromosomes Cancer 48: 340–350. 10. Brieger A, Adam R, Passmann S, Plotz G, Zeuzem S, et al. (2011) A CRM1- dependent nuclear export pathway is involved in the regulation of MutLalpha subcellular localization. Genes Chromosomes Cancer 50: 59–70. 11. Brieger A, Plotz G, Raedle J, Weber N, Baum W, et al. (2005) Characterization of the nuclear import of human MutLalpha. Mol Carcinog 43: 51–58. 12. Raevaara TE, Gerdes AM, Lonnqvist KE, Tybjaerg-Hansen A, Abdel- Rahman WM, et al. (2004) HNPCC mutation MLH1 P648S makes the functional protein unstable, and homozygosity predisposes to mild neurofibro- matosis type 1. Genes Chromosomes Cancer 40: 261–265. 13. Raevaara TE, Korhonen MK, Lohi H, Hampel H, Lynch E, et al. (2005) Functional significance and clinical phenotype of nontruncating mismatch repair variants of MLH1. Gastroenterology 129: 537–549. 14. Raevaara TE, Vaccaro C, Abdel-Rahman WM, Mocetti E, Bala S, et al. (2003) Pathogenicity of the hereditary colorectal cancer mutation hMLH1 del616 linked to shortage of the functional protein. Gastroenterology 125: 501–509. Fluorescent Tags Can Impair DNA Mismatch Repair successful. Restriction digests were separated on 2% agarose gels, stained with ethidium bromide and bands were quantified using Quantity One Software v4.6.1 (Bio-Rad, Hercules, CA, USA). Repair efficiency of unlabeled MutLa was set at 100 percent and repair of fluorescent tagged MutLa was determined in relation to the wild-type sample that was expressed, processed and tested in parallel. Although the amount of mismatched plasmid DNA present in parallel incubations is always identical (aliquoted from one master mix), phenol-extraction/ethanol precipitation of the processed plasmid can show differences in recovery, and therefore the overall DNA amounts can differ from lane to lane. However, repair efficiency is measured as quotient of the intensities of bands indicating repair and the sum of all band intensities and therefore gives an accurate repair value independent of the amount of DNA actually recovered during plasmid extraction. All experiments were performed in quadruplicate. Structural modeling of fluorescent labeled MutLa Structural modeling of MutLa as well as three-dimensional structures of GFP- or Red-proteins has been described previously [24,25,26,27]. 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Biochem Biophys Res Commun 340: 852–859. 24. Kosinski J, Plotz G, Guarne A, Bujnicki JM, Friedhoff P (2008) The PMS2 subunit of human MutLalpha contains a metal ion binding domain of the iron- dependent repressor protein family. J Mol Biol 382: 610–627. 25. Plotz G, Welsch C, Giron-Monzon L, Friedhoff P, Albrecht M, et al. (2006) Mutations in the MutSalpha interaction interface of MLH1 can abolish DNA mismatch repair. Nucleic Acids Res 34: 6574–6586. 26. Guarne A, Ramon-Maiques S, Wolff EM, Ghirlando R, Hu X, et al. (2004) Structure of the MutL C-terminal domain: a model of intact MutL and its roles in mismatch repair. EMBO J 23: 4134–4145. 27. Guarne A, Junop MS, Yang W (2001) Structure and function of the N-terminal 40 kDa fragment of human PMS2: a monomeric GHL ATPase. EMBO J 20: 5521–5531. 28. Kosinski J, Hinrichsen I, Bujnicki JM, Friedhoff P, Plotz G (2010) Identification of Lynch syndrome mutations in the MLH1-PMS2 interface that disturb dimerization and mismatch repair. Hum Mutat 31: 975–982. PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31863