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. 2023 Dec 18;24(24):17619.
doi: 10.3390/ijms242417619.

Mismatch Repair Protein Msh6Tt Is Necessary for Nuclear Division and Gametogenesis in Tetrahymena thermophila

Lin Wang  1 , Sitong Yang  1 , Yuhuan Xue  1 , Tao Bo  1   2 , Jing Xu  1   3 , Wei Wang  1   2
Affiliations

Mismatch Repair Protein Msh6Tt Is Necessary for Nuclear Division and Gametogenesis in Tetrahymena thermophila

Lin Wang et al. Int J Mol Sci. .

Abstract

DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in E. coli or its eukaryotic homolog, the MutSα (Msh2-Msh6) complex, recognizes base mismatches and initiates the mismatch repair mechanism. Msh6 is an essential protein for assembling the heterodimeric complex. However, the function of the Msh6 subunit remains elusive. Tetrahymena undergoes multiple DNA replication and nuclear division processes, including mitosis, amitosis, and meiosis. Here, we found that Msh6Tt localized in the macronucleus (MAC) and the micronucleus (MIC) during the vegetative growth stage and starvation. During the conjugation stage, Msh6Tt only localized in MICs and newly developing MACs. MSH6Tt knockout led to aberrant nuclear division during vegetative growth. The MSH6TtKO mutants were resistant to treatment with the DNA alkylating agent methyl methanesulfonate (MMS) compared to wild type cells. MSH6Tt knockout affected micronuclear meiosis and gametogenesis during the conjugation stage. Furthermore, Msh6Tt interacted with Msh2Tt and MMR-independent factors. Downregulation of MSH2Tt expression affected the stability of Msh6Tt. In addition, MSH6Tt knockout led to the upregulated expression of several MSH6Tt homologs at different developmental stages. Msh6Tt is involved in macronuclear amitosis, micronuclear mitosis, micronuclear meiosis, and gametogenesis in Tetrahymena.

Keywords: Tetrahymena thermophila; gametogenesis; mismatch repair protein Msh6Tt; nuclear division.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterization of MSH6Tt in T. thermophila. (A) Msh6Tt is colored from domain I to domain VI with red, yellow, green, cyan, purple-blue, and hot pink. (a), The complete modeling of Msh6Tt entire protein using Phyre 2 with intensive modeling mode. (b), Four views of Msh6Tt related to 90° rotations as indicated. (c), Two adjacent F-X-E motifs in domain I. Structural domain I is shown as red lines, and the F-X-E motifs are shown as blue–white sticks. The amino acid positions of the two F-X-E motifs are 291–293 and 295–297. (B) The relative expression profiles of MSH6Tt at different development stages. The expression of MSH6Tt was analyzed by qRT-PCR. The y-axis indicates the relative expression of MSH6Tt. The data were normalized to expression of MSH6Tt at 6 h of starvation. For growing cells, i, m, and h correspond to ~1 × 105 cells/mL, ~3.5 × 105 cells/mL, and ~1 × 106 cells/mL, respectively. For starvation, ~2 × 105 cells/mL were collected at 0, 6, 12, and 24 h, referred to as starvation (h) 0, 6, 12, and 24. For conjugation, equal volumes of B2086 and CU428 cells were mixed, and samples were collected at 0, 2, 4, 6, 8, 10, 12, 16, and 18 h after mixing, referred to as conjugation (h) 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18.
Figure 2
Figure 2
The localization of Msh6Tt-3HA in the amitotic MAC and mitotic MIC during vegetative proliferation and starvation stages. (A) Schematic representation of generating recombinant Msh6Tt-3HA mutants in T. thermophila. The pink arrow in the wild type (WT) locus indicates the MSH6Tt. The pink arrow and yellow box present in the targeting construct indicate the homologous arm. The 1415 bp green box is replaced by 3HA and Neo4 cassette. The black arrows indicate the position of the PCR primers when identifying the mutant cell line (the length of the arrow is independent of the primer length). (B) The identification of Msh6Tt-3HA-B2086 and Msh6Tt-3HA-CU428 mutants. M indicates marker; 1–3 indicate Msh6Tt-3HA-CU428 mutants; 4 and 8 indicate WT cells; 5–7 indicate Msh6Tt-3HA-B2086 mutants. The WT gene and mutation sites were amplified by PCR, which should be 2633 bp for the mutation site and 1415 bp for the WT gene. The black arrows in the figure (A) mark the positions of the identification primers in this PCR. (C) (a–f), Immunofluorescence localization of Msh6Tt-3HA in the amitotic MAC and mitotic MIC during vegetative proliferation. (g), WT negative control; (a’,b’) are five times the corresponding parts of a and b, respectively. The large white arrows indicate the MACs; the small white arrows indicate the MICs; the yellow arrow indicates that Msh6Tt-3HA forms a dumbbell shape and is localized in the circular region and the middle part. # indicates the new MICs after mitosis; * indicates the new MACs after amitosis. Hollow pink arrows indicate the localization of Msh6Tt-3HA in the perinuclear region of the MIC. The solid pink arrow indicates the localization of Msh6Tt-3HA in the MIC. (D) Immunofluorescence localization of Msh6Tt-3HA during starvation. The indirect immunofluorescence localization signal of Msh6Tt-3HA is green (HA label). DAPI staining of the nuclei is blue. The large white arrows indicate the MACs; the small white arrows indicate the MICs. The scale bar is 10 μm.
Figure 3
Figure 3
The localization of Msh6Tt-3HA in the MIC and new developing MAC during conjugation stage. (A) (a–h), Immunofluorescence localization of Msh6Tt-3HA during conjugation. (a’–d’) is five times the corresponding part (marked with a yellow box) of (a–d). (B) Msh6Tt-3HA localized on chromatin in spread cells. The chromatin-binding proteins will remain in the nucleus after using an enhanced detergent to spread cells. The detergent treatment disrupts non-specific interactions and removes loosely bound proteins from the chromatin. The large white arrows indicate the MACs; the small white arrows indicate the MICs. # indicates the new MICs; * indicates the new MACs. The indirect immunofluorescence localization signal of Msh6Tt-3HA is green (HA label). The DAPI staining of the nuclei is blue. The scale bar is 10 μm.
Figure 4
Figure 4
Knockout of MSH6Tt resulted in abnormal nuclear divisions in Tetrahymena during the vegetative growth stage. (A) Schematic representation for generating recombinant MSH6TtKO mutants in T. thermophila. The blue arrow in the WT locus indicates the MSH6Tt gene; the yellow boxes with the black diagonal filling in the WT locus and targeting construct indicate the homologous arms. The MSH6Tt cassette is replaced by the Neo4 cassette at the mutated locus. The black arrows indicate the position of the PCR primer when identifying the mutant cell line (the length of the arrow is independent of the primer length). (B) The identification of MSH6TtKO-B2086 and MSH6TtKO-CU428 mutants. M indicates marker; 1–3 indicate MSH6TtKO-CU428 mutants; 4 indicates WT cells; 5–7 indicate MSH6TtKO-B2086 mutants. The WT and mutation sites were amplified by PCR; 2630 bp is for the mutation site, and 4327 bp is for the WT site. The black arrows in the figure (A) mark the positions of the primers. (C) MSH6Tt knockout affected nuclear division during vegetative growth. (1–10), The abnormal nuclear division of MSH6TtKO mutants. The topmost part of the diagram shows normal nuclear development during the vegetative stage of WT in T. thermophila. The large white arrows indicate the MACs; the small white arrows indicate the MICs. # indicates the new MICs after mitosis; * indicates the new MACs after amitosis. DAPI staining of the nuclei is blue. The scale bar is 10 μm.
Figure 5
Figure 5
Abnormal nuclear divisions in the MSH6TtKO mutant cell line during the conjugation stage. (A) The nuclear development during the conjugation stage in T. thermophila. (1), crescent elongating of MIC; (2), the first meiotic division (Meiosis Ι); (3), the second meiotic division (Meiosis II); (4), MIC “selection”; (5), Exconjugant with two MACs and one MIC. The topmost part of the diagram shows a diagram of the cell development model during the conjugation in T. thermophila. (B) Abnormal nuclear divisions in the MSH6TtKO mutants during the conjugation stage. (a), crescent elongating of MIC; (b,c), MICs in MSH6TtKO mutants were fragmented at the end of the first meiotic division; (d–f), meiotic products were stacked at the posterior of the cell; (g,h), meiotic products were stacked at the anterior of the cell; (i,j), abnormal single cells with multiple MICs (i) and without MICs (j). DAPI stains the nuclei. The scale bar is 10 μm.
Figure 6
Figure 6
Msh6Tt-3HA interacts with both MMR-dependent factors and MMR-independent factors. (A) A protein interaction network map showing the proteins that interacted with Msh6Tt-3HA at 3 h of conjugation. The HA tag at the C-terminus of Msh6Tt-3HA was used to immunoprecipitate the Msh6Tt interaction proteins in T. thermophila. WT cell lysates, where there was no HA tag, were also used to immunoprecipitate, with the protein obtained being used as a blank control in subsequent analyses and subtracted from the experimental group. The interacting proteins were identified by mass spectrometry analysis. MaxQuant was used to analyze mass spectrometry data. (B) Protein–protein docking results for (a) Msh6Tt and Dmc1, (b) Msh6Tt and Rfc2, (c) Msh6Tt and TTHERM_00313630. (C) Msh6Tt and Msh2Tt have consistent and different interacting protein partners. (a) Msh2 Tt interacted with Msh6Tt-3HA at both 3 h and 8 h of conjugation. (b)The eight proteins interacted with both Msh6Tt-3HA and Msh2Tt-3HA at 3 h of conjugation. Msh2Tt-3HA protein interaction data were acquired from our previous data [34].
Figure 7
Figure 7
Expression of MSH2Tt maintains the stability of the Msh6Tt. (A) Knocking down MSH2Tt by adding Cd2+ in the msh2i mutants and WT mating pairs. The relative expression level of MSH2Tt (a) and MSH6Tt (b) in the mating pairs was detected by qPCR. The control was the gene expression level of MSH2Tt or MSH6Tt without Cd2+. Samples were collected at 4.5 h of conjugation. The Y-axis indicates the relative normalized expression of MSH2Tt (a) and MSH6Tt (b). Error bars represent the standard deviations for three replicates. * p < 0.05, ** p < 0.01. p values were calculated using Student’s t-test. (B) The expression level of Msh2Tt-3HA and Msh6Tt-3HA when the msh2i mutants mating with Msh2Tt-3HA or Msh6Tt-3HA mutants. (a), Western blot showed that the expression of Msh2Tt-3HA decreased after knocking down MSH2Tt by adding Cd2+ to the msh2i and Msh2Tt-3HA mating pairs. (b), The expression of Msh6Tt-3HA was not detected by Western blot after knocking down MSH2Tt by adding Cd2+ to the msh2i and Msh6Tt-3HA mating pairs. Samples were collected at 4.5 h of conjugation. The internal reference is Pcna. (C) The expression levels of MSH2Tt (a), MSH6L3Tt (b), MSH3Tt (c), and MSH3L1Tt (d) at different growth stages of Tetrahymena in WT cells and MSH6TtKO mutants. L, vegetative growing cells; S, starvation cells; equal volumes of B2086 and CU428 cells were mixed, and samples were collected at 2 h and 8 h after mixing, referred to as C-2 and C-8. The Y-axis indicates the relative normalized expression of MSH2Tt (a), MSH6L3Tt (b), MSH3Tt (c), and MSH3L1Tt (d). Error bars represent the standard deviations for three replicates. ** p < 0.01, *** p < 0.001. p values were calculated using Student’s t-test.
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References

    1. Boland C.R., Luciani M.G., Gasche C., Goel A. Infection, inflammation, and gastrointestinal cancer. Gut. 2005;54:1321–1331. doi: 10.1136/gut.2004.060079. - DOI - PMC - PubMed
    1. McCulloch S.D., Kunkel T.A. The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases. Cell Res. 2008;18:148–161. doi: 10.1038/cr.2008.4. - DOI - PMC - PubMed
    1. Shimizu M., Gruz P., Kamiya H., Kim S.R., Pisani F.M., Masutani C., Kanke Y., Harashima H., Hanaoka F., Nohmi T. Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases. EMBO Rep. 2003;4:269–273. doi: 10.1038/sj.embor.embor765. - DOI - PMC - PubMed
    1. Kunkel T.A. Evolving views of DNA replication (in)fidelity. Cold Spring Harb. Symp. Quant. Biol. 2009;74:91–101. doi: 10.1101/sqb.2009.74.027. - DOI - PMC - PubMed
    1. Hoeijmakers J.H. DNA damage, aging, and cancer. N. Engl. J. Med. 2009;361:1475–1485. doi: 10.1056/NEJMra0804615. - DOI - PubMed

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