At the very center of sexual reproduction is meiosis. During meiosis, the formation of meiotic Double-Strand-Breaks (DBSs) and their repair by homologous recombination are widely conserved events occurring among most eukaryote species. Meiosis-specific DSB formation requires at least nine proteins (Spo11, Ski8, Rec102, Rec104, Mei4, Mer2, Rec114, Mre11/Rad50/Xrs2) in S. cerevisiae, and the resection of the DSB ends requires additional four proteins (Mre11/Rad50/Xrs2, and Sae2). Spo11 has been identified as the catalytic component of this DSB-initiating complex. However, the roles played by the majority of these proteins are not clear. I have purified the recombinant Spo11/Ski8/Rec102/Rec104 complex, characterized its DNA binding ability as well as its cleavage activity on supercoiled plasmid DNA.
Sae2 functions in both meiotic and mitotic repair of DNA double-strand breaks (DSBs) in S. cerevisiae. In vivo experiments have shown that Sae2 collaborates with the Mre11/Rad50/Xrs2 (MRX) complex in DNA end processing. Our laboratory previously showed that recombinant Sae2 exhibits endonuclease activity on single-stranded DNA and single-strand/double-strand DNA junctions using purified proteins in vitro. The MRX complex stimulates Sae2 endonuclease activity on single-stranded DNA close to single-strand/double-strand junctions, through its endonucleolytic activity. However, Sae2 contains no conserved typical nuclease domain, and it only shares very limited homology with its human functional counterpart CtIP. To characterize Sae2 and the active sites responsible for its nuclease activity, I used partial proteolysis and site-directed mutagenesis to analyze the protein. Biochemical assays in vitro show that acidic residues in the central domain play an important role in Sae2 endonuclease activity. Sae2 has also been shown to be phosphorylated by CDK (Cyclin-Dependent Kinase) during the S and G2 phases of the cell cycle, as well as by Tel1/Mec1 upon DNA damage. These modifications are essential for the function of Sae2 in DNA repair, but the function of these modifications are not clear. I have demonstrated that, in the presence of MRX, Sae2 (5D/S267E) mimicking constitutive phosphorylation by CDK and Mec1/Tel1 can assist the 5’ to 3’ exonuclease Exo1 significantly in 5’ end resection by suppressing the inhibitory effect of Ku. These results suggest that Sae2 is a critical switching protein which determines the choice between HR and NHEJ in yeast cells upon DNA damage. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/ETD-UT-2011-08-4169 |
| Date | 15 January 2013 |
| Creators | Shen, Mingjuan |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Type | thesis |
| Format | application/pdf |
Page generated in 0.0024 seconds