Fluorescence detection of DNA mismatch repair in human cells

Mismatched base pairs, produced by nucleotide misincorporation by DNA polymerase, are repaired by the mismatch repair (MMR) pathway to maintain genetic integrity. We have developed a method for the fluorescence detection of the cellular MMR ability. A mismatch, which would generate a stop codon in the mRNA transcript unless it was repaired, was introduced into the gene encoding the enhanced green fluorescent protein (EGFP) in an expression plasmid. When MMR-proficient HeLa cells were transformed with this plasmid, the production of active EGFP was observed by fluorescence microscopy. It was assumed that the nick required to initiate the MMR pathway was produced non-specifically in the cells. In contrast, fluorescence was not detected for three types of MMR-deficient cells, LoVo, HCT116, and DLD-1, transformed with the same plasmid. In addition, the expression of a red fluorescent protein gene was utilized to avoid false-negative results. This simple fluorescence method may improve the detection of repair defects, as a biomarker for cancer diagnosis and therapy.

Defects in DNA repair pathways cause diseases, and in the case of MMR, Lynch syndrome 14,15 , which was previously referred to as hereditary non-polyposis colorectal cancer, is a typical example. It is the most common inherited colorectal cancer syndrome, and is caused by germline mutations in one of the four MMR genes, MLH1, MSH2, MSH6, and PMS2. A somatic mutation of the wild-type allele results in the loss of the MMR function, and leads to colorectal cancer, as well as endometrial cancer. Since Lynch syndrome is an autosomal dominant disorder, its diagnosis is important, and surveillance is required for family members of patients diagnosed with this syndrome. To detect the MMR deficiency, the microsatellite instability (MSI) is analyzed 16,17 . The alterations in the lengths of simple repeat sequences, called microsatellites, correlate with the MMR function, and according to the ratios of the length alterations found in the analyzed microsatellites, the results are classified into three categories, MSI-H (MSI-high), MSI-L (MSI-low), and MSS (microsatellite stable) 18 . The MSI-H phenotype strongly supports the Lynch syndrome diagnosis. An alternative method for the screening of Lynch syndrome is the analysis of the MMR proteins by immunohistochemistry (IHC). However, neither of these tests can definitively detect the MMR deficiency 17 . For example, the majority of tumors from MSH6-mutation carriers do not have MSI-H 19 , and the complex relationship between MMR defects and MSI patterns has also been noted 20 . In IHC, false-positive results may be obtained for dysfunctional MMR gene products containing a single amino acid substitution, particularly outside the dimerization domains 21 .
In this study, we developed a method to detect the cellular MMR ability by fluorescence. We previously reported the fluorescence-mediated detection of the base excision repair (BER) 22 and nucleotide excision repair (NER) 23 of DNA lesions induced by oxidative stress and UV irradiation, respectively, in cultured human cells (Fig. 1a,b). In both cases, a fluorophore-quencher pair, which was originally used in a molecular beacon 24 , was incorporated into a hairpin-shaped oligonucleotide or a plasmid containing a damaged base. In the present study, the expression of the gene encoding green fluorescent protein, containing a mismatch, was utilized to detect the overall process of MMR (Fig. 1c).

Results
Preparation of the expression plasmid containing a mismatch. In this study, we intended to develop a system in which a mismatch-containing gene encoding a full-length fluorescent protein is expressed in cells only when it is repaired, to detect the cellular MMR ability. We constructed a plasmid (pBSII EGFP) to express the gene encoding the enhanced green fluorescent protein (EGFP), which has the F64L and S65T substitutions in the original green fluorescent protein 25 , and then introduced a C/A mismatch at the first position of the codon for Q157 (CAG) of this protein (pBSII EGFP C/A, Fig. 1c). The T/G mismatch was avoided because it could be recognized by other enzymes, such as the methyl-CpG domain protein and thymine DNA glycosylase 26 , and the C/A mismatch is a good substrate for the MMR pathway 27 . Since the template strand had the sequence 3′-ATC-5′,  22 . The probe contained an oxidatively damaged base in the center and a fluorophore-quencher pair at the end, and the phosphodiester linkages were changed to nuclease-resistant phosphorothioate analogs in the regions that were not required for enzyme binding. Fluorescence was detected when the probe was cleaved by NTH1 in human cells. (b) Detection of nucleotide excision repair developed in our previous study 23 . A plasmid was used as a scaffold, and the fluorophore and the quencher were attached to the base moieties near a UV-induced lesion. Fluorescence was detected when the dual-incision product was degraded in cells. (c) Detection of MMR in this study. Cells are transformed with a plasmid containing a mismatch (shown in red) in the EGFP gene, which is expressed under the control of the CMV promoter. In the absence of MMR, the mRNA transcript contains a stop codon, UAG. However, when the cells are MMR-proficient, the repair of the bottom strand results in the generation of the original EGFP gene, and fluorescence is detected in the cells. We expected that the initial nick required for MMR would be produced in the cells in a random manner. There is a possibility that the top strand is repaired, but the full-length EGFP is not produced in this case.
Scientific RepoRTS | (2018) 8:12181 | DOI:10.1038/s41598-018-30733-x the mRNA produced under the control of the cytomegalovirus (CMV) promoter in the cells transformed with this plasmid would contain a stop codon, UAG, in the absence of the cellular MMR activity. In MMR-proficient cells, we expected that two types of plasmids would be generated. One of them should contain the C·G pair at the first position of this codon, produced by the removal of the A in the bottom strand to be used as the template in transcription, as shown in Fig. 1c. Since the CAG sequence at this site in the mRNA produced from the repaired plasmid is the codon for glutamine, the full-length EGFP would be produced. In other words, fluorescence would be observed, depending on the cellular MMR ability. An expression plasmid containing the normal EGFP gene without the mismatch (pBSII EGFP C·G) was also prepared in the same manner.
In order to initiate the MMR pathway, not only a mismatch but also a nick is required in the DNA, as described in the Introduction. At the beginning of this study, we planned to either incorporate a ribonucleotide 11,12 into the mismatch-containing primer used for the preparation of the double-stranded plasmid from the single-stranded DNA, or introduce a nick into the plasmid with a nicking endonuclease 28 . However, we previously found that the plasmids were degraded in the cells within several hours during the culture at 37 °C 23 . Therefore, we decided to test the plasmids without the nick, and expected that a nick would be produced non-specifically in the cells.

Transformation of MMR-proficient and MMR-deficient cells. To begin with, HeLa and LoVo cells,
which are proficient and deficient in MMR, respectively, were transformed with pBSII EGFP C·G containing the normal EGFP gene, and the production of the fluorescent protein was confirmed by observing the cells with a fluorescence microscope. In these experiments, an overnight culture after the transformation was required to obtain sufficient fluorescence intensities ( Supplementary Fig. S1). Next, the cells were transformed with pBSII EGFP C/A, which contained the C/A mismatch in the EGFP gene. As the MMR-deficient cells, we also used HCT116 and DLD-1, which are human colon cancer cell lines similar to the LoVo cells, but derived from different origins 29 . Fluorescence was detected in the HeLa cells transformed with this plasmid (Fig. 2a), and the quantitative analysis of the micrographic images revealed that the fluorescence intensity was about one-half of that observed for the same cells transformed with pBSII EGFP C·G (Fig. 2b). In contrast, fluorescence was hardly visible in all of the MMR-deficient cell types (Fig. 2). The experiments were repeated at least three times, and the results were quite reproducible.
Confirmation of transformation using the tdTomato gene expression. Since fluorescence is detected only when the correct EGFP gene is expressed, there is no possibility of obtaining false-positive results in this method. However, a failure in transformation results in false-negative observations, in which the fluorescence from MMR-proficient cells is not detected. To avoid such errors, we tried to improve the method. A gene encoding a red fluorescent protein, tdTomato 30 , which is transcribed from the CMV promoter, was inserted into the EGFP gene expression plasmid, as shown in Fig. 3a. In practice, pBSII EGFP-tdTomato was prepared by ligating the PCR product containing the CMV promoter and the tdTomato gene with the large fragment of pBSII EGFP (Fig. 4). Subsequently, the C/A mismatch was produced in the EGFP gene in the same manner as in pBSII EGFP  Fig. 3a) and the counterpart without the mismatch (pBSII EGFP-tdTomato C·G), with the expectations that the red fluorescence of tdTomato would be detected when the transformation is successful, while the green fluorescence of EGFP would indicate the cellular MMR ability (Fig. 3b). After an incubation at 37 °C for 24 h, the transformed cells were observed with a fluorescence microscope at two wavelengths (Fig. 5a). As expected, the red fluorescence was detected regardless of the cellular MMR ability, and the expression of the repaired EGFP gene was observed only in the MMR-proficient HeLa cells. Although there was an obvious difference in the green fluorescence between the HeLa and LoVo cells transformed with pBSII EGFP-tdTomato C/A, the number of the green fluorescent HeLa cells was considerably smaller than that observed for the positive control (pBSII EGFP-tdTomato C·G) (Fig. 5b).
To obtain more information, the cells were analyzed by flow cytometry (Fig. 6 and Supplementary Fig. S2). When the HeLa cells were transformed with pBSII EGFP-tdTomato C·G, a significant correlation was observed between the fluorescence intensities of EGFP and tdTomato because the two genes were designed to be coexpressed in each cell. However, in the experiment using pBSII EGFP-tdTomato C/A, about 30% of the cells did not show the EGFP fluorescence, probably due to the mismatch remaining in the EGFP gene, and the fluorescence intensity of EGFP was relatively low.

Discussion
In this study, we have developed a method for the fluorescence detection of the cellular MMR ability, using the EGFP gene expression system. In the MMR-proficient cells, the normal EGFP gene is generated by the repair of the mismatch, as shown in Fig. 1c, whereas the stop codon in the mRNA transcribed from the mismatch-containing gene inhibits the production of the full-length EGFP, if MMR does not function in the cells. There were two concerns at the beginning of this study. One was whether a plan to introduce a nick to initiate the MMR pathway should be executed, and the other was whether replication, which would produce the normal EGFP gene, would occur in the cultured cells. Judging by the results obtained in this study, neither of these two points needed to be considered. It was assumed that the nick was produced by a cellular nuclease, as expected from our previous study 23 , and the replication was avoided by using the pBlueScript vector, which lacks the eukaryotic replication origin. This study is the first visualization of in vivo MMR, and it is important that the correlation of the fluorescence detection with the mismatch correction was demonstrated experimentally. The method described here may be applied to Lynch syndrome screening. The difference between this method and the MSI and IHC tests, which are currently used for Lynch syndrome screening, is that the entire process of MMR is monitored directly in this method, and there is no possibility of obtaining false-positive results because fluorescence is detected only when MMR is completed. False-negative results can be avoided by using the pBSII EGFP-tdTomato system (Fig. 3). In the experiments using pBSII EGFP-tdTomato C/A, the ratio of the HeLa cells showing the green fluorescence was lower (Fig. 5b) than that observed for the HeLa cells transformed with pBSII EGFP C/A (Fig. 2b), and the reproducibility of the results was confirmed by changing the experimenter. This may be because pBSII EGFP-tdTomato C/A (6661 bp) is longer than pBSII EGFP C/A (4414 bp). Since the plasmid is randomly nicked in the transformed cells, the particular nick at a position appropriate for the initiation of MMR may be formed at a lower frequency when the plasmid size is large. More information was obtained from the results of the flow cytometry analysis (Fig. 6). In the case of the cells transformed with pBSII EGFP-tdTomato C/A, about 30% of the cells were found in the EGFP-negative region (shown in red), and even in the EGFP-positive region, the fluorescence intensity was lower than that observed for the cells transformed with pBSII EGFP-tdTomato C·G. Not only the unrepaired plasmid but also this low intensity is thought to be the reason for the reduced fluorescence intensity in the quantification of the microscopy images (Fig. 5b).
In addition to Lynch syndrome, there are several other aspects where MMR is important in cancer treatment. Recently, immune checkpoint inhibitors have attracted attention in cancer immunotherapy. Activated T cells, which induce apoptosis of the target cancer cells, have an immune checkpoint protein called programmed cell death 1 (PD-1) on their surfaces, and the binding of one of the two ligands for this protein (PD-L1 and PD-L2) expressed on the surfaces of the cancer cells to PD-1 inhibits the cytotoxic activity of the T cells 31 . Therefore, antibodies to PD-1 can maintain the anticancer immune response of the T cells 32 . However, the clinical benefit of an immune checkpoint blockade with pembrolizumab, a monoclonal antibody to PD-1, reportedly depends on the MMR status, and MMR-deficient tumors are more responsive to the PD-1 blockade 33 . Moreover, adjuvant chemotherapy with fluorouracil also depends on the MMR activity. The treatment improved the overall survival among the patients with MSS and MSI-L tumors, but there was no benefit in the MSI-H group 34 .
As described above, the importance of analyzing the MMR status of cells is increasing. The simple fluorescence method developed in this study will contribute to many research fields ranging from biology to clinical studies, and in particular may improve biomarker-driven personalized diagnosis and therapy for cancer patients.

Methods
Preparation of pBSII EGFP. The DNA fragment containing the CMV promoter, the EGFP gene, and the   and 535 nm, respectively, and the same microscopic fields were observed at 605 nm with excitation at 540 nm to detect tdTomato. HeLa and LoVo cells were used as MMR-proficient and deficient cells, respectively. (b) Comparison of the EGFP fluorescence normalized by tdTomato. The fluorescence intensities were quantified using the BZII-analyzer software, and the EGFP/tdTomato ratios were compared between the C·G-and C/Acontaining plasmids.  Fig. S3a), the product was treated with BssHII (New England Biolabs, 5 units) in buffer (10 μl) containing 20 mM Tris-acetate (pH 7.9), 50 mM potassium acetate, 10 mM magnesium acetate, and 100 μg/ml BSA at 50 °C for 1 h. pBlueScript II SK (−) (Agilent Technologies, 2.8 μg) was treated with BssHII (5 units) and Antarctic phosphatase (New England Biolabs, 5 units) in the above buffer (30 μl) at 50 °C for 1 h. The BssHII-treated plasmid and the PCR product were precipitated with ethanol, and dissolved in water (25 μl and 10 μl, respectively). Aliquots of these solutions (1 μl and 3 μl, respectively) were mixed, and after the addition of DNA ligation mix (Takara Bio, 4 μl), the mixture was incubated at room temperature for 30 min. Escherichia coli DH5α competent cells (Takara Bio, 50 μl) were mixed with the ligation mixture (4 μl) on ice, and cultured on Luria-Bertani (LB) agar plates with ampicillin overnight. Single colonies were isolated and cultured in LB medium with 50 μg/ml ampicillin (LB Amp) (1 ml) for 6 h. The DNA in each colony was purified with the QIAprep Spin Miniprep Kit (Qiagen) and analyzed by gel electrophoresis after BssHII treatment (Supplementary Fig. S3b). The correct insertion of the PCR product into the plasmid was confirmed by the sequence analysis.
Flow cytometry. HeLa S3 cells were seeded into a 12-well plate, grown to approximately 80% confluence, and transfected with plasmids (2.4 μg per well) using Lipofectamine 2000 (5 μl per well). After 24 h, cells were harvested and resuspended into D-PBS (Nacalai Tesque) plus 2% FBS, and the suspension was passed through a cell strainer. The obtained cell suspensions were analyzed by CytoFLEX S flow cytometer (Beckman Coulter), in which green (excitation at 488 nm) and yellow (excitation at 561 nm) fluorescence channels were calibrated with HeLa cells transformed with pBSII EGFP and pCMV-tdTomato plasmids, respectively. After gating for debris exclusion and doublet discrimination, 10,000 single cells were analyzed. The data were processed with the CytExpert software (Beckman Coulter).