The functions of the MSH2 and MLH1 proteins during meiosis in Tetrahymena thermophila

• Main Author: Sun, Lin
• Other Authors: Cupples, Claire G.
• Language: English; en
• Published: 2009
• Subjects: DNA mismatch repair; Meiosis; MSH2 and MLH1; Tetrahymena thermophila; UVic Subject Index::Sciences and Engineering
• Online Access: http://hdl.handle.net/1828/1720

Msh2 and Mlh1 proteins from Tetrahymena thermophla are homologues of MutS and MutL from Escherichia coli respectively. MutS and MutL are DNA mismatch repair proteins. In eukaryotes, MutS homologues recognize the replication errors and MutL homologues interact with MutS homologues and other proteins to make the repair occur. Biolistic transformation has been done to make the msh2 and mlh1 single knockouts in the macronuclei of different strains and the knockouts were verified complete. Two strains of WT crossing KO or KO crossing KO, with different mating types, were induced to conjugate. The processes were studied by microscopy using DAPI staining. For the msh2 knockouts, there were no crescent micronuclei formed throughout the conjugation of two knockout cells, and the pairing level was reduced severely. However, a knockout cell and a wild-type cell could conjugate normally at a high level pairing efficiency. Msh2 protein seems to be important to cell pairing and indispensible for the formation of the crescent micronuclei during cell conjugation. For the mlh1 knockouts, the pairing level of a knockout and a wild-type was reduced by half and the pairing level of two knockouts was reduced more than 80%; however, the paired cells in both could complete the conjugation with delay. Pms2 protein may have redundant roles in the MutL heterodimer (Mlh1-Pms2). In addition, chemical mutagens treated knockout was crossed with non-treated wild-type and the conjugation was compared with treated wild-types. Most of the treated knockout cells could not pair after starvation and mixing with non-treated wild-type cells, which means most of the cells could not enter meiotic phase. It is probable that G2/M checkpoint arrested the meiotic cell cycle and the intra-S phase was inactivated. Thus, Msh2 protein may have a role in the meiotic intra-S phase checkpoint system.