In this thesis, we aimed to identify HJ resolvases through protein sequence alignment. We identified <i>Saccharomyces cerevisiae</i> Yol093wp by the PSI-BLAST search with the sequence of the yeast mitochondrial resolvase Cce1p or Ydc2p. Yol093wp homologs are widely found in eukaryotes. These homologs of the Yol093wp family are poorly related to each other and have small protein sizes; similar to those observed among the families of known resolvases. In this work, a variety of general and specific approaches were used to characterize the functions of <i>YOL093W</i>. We have shown that His-tagged Yol093wp expressed and enriched from <i>E. coli</i>, and the Yol093wp extracts might have DNA binding properties. Interestingly, <i>in vitro</i> studies revealed the preferential binding of Yol093wp extracts to a mobile four-way junction than to a 60 nt-ssDNA or a 60 bp-dsDNA. The basis of this binding activity is still unclear. Further analysis will be required to elucidate the specificity of this DNA binding. Cells lacking Yol093wp have no obvious growth defects under standard growth conditions. Meiotic recombination assays showed that <i>YOL093W </i>deletion has not effect on meiotic recombination. A yeast two-hybrid screen and TAP tag purification were utilised to identify Yol093wp-interacting proteins. In the two-hybrid screen, only one protein, Mtl1p, was retrieved with relatively low significance. The TAP purification of Yol093wp complex showed that the Yol093w protein was synthesized in the TAP-tagged strain, and could be purified followed the TAP purification procedure. However, the identities of the potential interacting proteins identified did not throw much light on the function of Yol093wp. Finally, the immunofluorescence micrographs of Yol093wp-GFP and Yol093wp-13myc in conjunction with DAPI straining suggested that the Yol093w fusion proteins are located in the nucleus. All of these data may be indicative of the function of Yol093wp.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:642735 |
| Date | January 2004 |
| Creators | Chang, Meng-Ya |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13357 |
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