C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins Angela Brieger*, Guido Plotz, Inga Hinrichsen, Sandra Passmann, Ronja Adam, Stefan Zeuzem Department of Medicine I, University of Frankfurt/M., Frankfurt, Germany Abstract The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLa (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLa’s functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLa. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLa. In contrast, N-terminal tagged MutLas retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency. Citation: Brieger A, Plotz G, Hinrichsen I, Passmann S, Adam R, et al. (2012) C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins. PLoS ONE 7(2): e31863. doi:10.1371/journal.pone.0031863 Editor: Michael R. Volkert, University of Massachusetts Medical School, United States of America Received November 28, 2011; Accepted January 13, 2012; Published February 14, 2012 Copyright: 2012 Brieger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grant 2005.085.2 of the Wilhelm Sander-Stiftung, grant 109651 of the Deutsche Krebsstiftung and institutional funds of the Goethe-University Frankfurt. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: a.brieger@em.uni-frankfurt.de Introduction DNA mismatch repair (MMR) is responsible for the correction of DNA replications errors and therefore essential for maintaining genomic stability and preventing tumor formation. Germline mutations in any of four MMR genes (MLH1, MSH2, MSH6, PMS2) cause the development of Lynch syndrome. MMR deficiency in tumors of patients with Lynch syndrome is characterized by microsatellite instability (MSI), which can be detected in more than 95% of associated carcinoma [1]. The initial step of human MMR is the recognition of mismatches by the heterodimer MutSa (MSH2 and MHS6) or MutSb (MSH2 and MSH3). In the subsequent step the MutS complex recruits a MutL complex, most dominantly MutLa (MLH1 and PMS2) of which MLH1 is the essential component stabilizing its dimeric partner protein PMS2. Especially the last C-terminal alpha helix of MLH1 seems to be most important for PMS2 stabilization but also for the correction of mispairs as well as for checkpoint signaling in response e.g. to 6-thioguanine [2]. Beside this, MutLa is responsible for the recruitment of many downstream proteins essential for the repair process. Moreover, several different MMR dependent but also independent proteins have been published to interact with MutLa [3,4,5,6,7], suggesting its involvement in several other cellular processes like apoptosis and protein transport. Therefore, binding capacities as well as accessibility of MutLa’s surface are supposed to be of utmost importance for the functional involvement of MutLa in these different processes. Many in vitro data have been published using N-terminal [8,9,10,11] or C-terminal [12,13,14,15,16,17] fluorescent tagged MMR proteins. However, fluorescent labeling might have significant influence on the functionality of tagged proteins [18,19,20]. Therefore, we investigated the influence of N- or C-terminal dye labeling of MutLa on expression level, cellular localization and repair function. Using different combinations of coexpressed GFP- and Red-labeled or unlabeled MLH1 and PMS2 proteins, we compared expression level, cellular localization and the MMR functionality of these MutLa variants with the untagged MutLa. Results Single expression of MLH1 or PMS2 is significantly influenced by fluorescent labeling In order to determine the influence of fluorescent labeling on single expressed MLH1 and PMS2 variants each of these proteins was transfected and expressed in HEK293T cells. As shown in Figure 1A, MLH1 is well expressed without coexpression of PMS2. However, N-terminal GFP (Figure 1A, lane 3) and C-terminal Red labeling (Figure 1A, lane 4) led to decreased expression levels. In contrast, PMS2 (Figure 1B), normally unstable without coexpressed heterodimeric partner protein MLH1 [2,22] and hardly expressed despite using overexpression-plasmid pcDNA3.1 (Figure 1B, lane 6), is well expressed and stable with N-terminal GFP or Red fluorescent labeling (Figure 1B, lane 7+9). However, C- terminal GFP or Red labeling resulted in very low or nearly undetectable expression of PMS2 (Figure 1B, lane 8+10), respectively. PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e31863