Investigation of the function of YOL093W in Saccharomyces cerevisiae

Resolution of four-way junction (Holliday junction, HJ) structures is a key event during recombination. The enzymatic activities that promote Holliday junction formation and resolution have been extensively characterized in bacteria. However, much less is known in eukaryotic cells and to identify similar activities in eukaryotes have been difficult. In this thesis, we aimed to identify HJ resolvases through protein sequence alignment. We identified Saccharomyces cerevisiae Yo1093wp by the PSIBLAST search with the sequence of the yeast mitochondrial resolvase Ccelp or Ydc2p. Yo1093wp homologs are widely found in eukaryotes. Each sequence of the Yo1093wp family has diverged substantially from every other with relatively small sizes, and contains some conserved acidic residues, which might contribute to the binding site catalytic metal ion(s). These are 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 YOL093W. We have shown that His-tagged Yo1093wp expressed in E. coli and has been enriched, and the Yo1093wp extracts might have DNA binding properties. Interestingly, in vitro studies revealed the preferential binding of Yo1093wp 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 Yo1093wp have no obvious growth defects under standard growth conditions. Meiotic recombination assays showed that YOL093W deletion has no effect on meiotic recombination. A yeast two-hybrid screen and TAP tag purification were utilised to identify Yo1093wp-interacting proteins. In the twohybrid screen, Mtllp that is involved in cell wall biogenesis was retrieved with relatively low significance. The TAP purification of Yo1093wp complex showed that the Yo1093w protein was synthesized in the TAP-tagged strain, and could be purified followed the TAP purification precedure. However, the identities of the potential interacting proteins identified did not throw much light on the function of Yol093wp. Finally, the immunofluorescence micrographs of Yo1093wp-GFP and Yo1093wp-13myc in conjunction with DAPI straining suggested that the Yo1093w fusion proteins are located in the nucleus. All of these data may be indicative of the function of Yo1093wp. Common Abbreviations aa Amino acid(s) Amp Ampicillin ARS Autonomous Replication Sequence ATP Adenosine 5'-triphosphate bp Base-pair Oc Degree Celsius cDNA Complementary Deoxyribonucleic Acid CEN Centromere Sequence cpm Counts per Minutes cm centimeter Da Dalton dATP Deoxyadenosine triphosphate dCTP Deoxycytidine tnphosphate dGTP Deoxyguanosine tnphosphate dfl'P Deoxythymine triphosphate DMSO Dimehtyl Sulfate DNA deoxyribonucleic acid DTT Dithiothreitol ECL Enhanced Chemi-Luminescence EDTA Ethylenediaminetetraacetic acid g gram(s) h hour(s) HA Haemagglutinin HA-i protein epitope HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid hph Hygromycin B HRP Horseradish Peroxidase kb kilobase kDa kilodalton kV kilovolt(s) L Litter(s) LB Luna Broth (medium) M Molar mA milli ampere(s) mg milligram(s) mM millimolar mmol millimole(s) mm minute(s) ml milliliter(s) mRNA Messenger Ribonucleic Acid MOPS 3-[N-Morpholino]-propanesulphonic acid MW Molecular Weight NP-40 Nonident P-40 detergent ng nanogram(s) nm nanometer(s) nt nucleotide 0D600 Optical Density at 600 nm ORF Open Reading Frame PAGE Polyacryamide Gel Electrophoresis PCR Polymerase Chain Reaction pmol picomole(s) RNA Ribonucleic Acid RT Room Temperature rpm Revolutions per minute SDS Sodium Dodecyl Sulphate TAE Tris/Acetate (buffer) TBS Tns-buffered Saline TEMIED N,N,N ',N '-tetramethyl-ethylenediamine tRNA transfer Ribonucleic acid Tns Tris(hydroxymethyl)aminomethane pg microgram(s) gI microliter(s) gm micrometer(s) U Unit(s) V Volt(s) v/v Volume per unit volume w/v Weight per unit volume YMM Yeast Minimal medium YPDA YeastlPeptoneIDextrose/Adenine (medium) Amino Acid Abbreviations Amino acid 3-letter code 1-letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartate Asp D Cystenine Cys C Glutamate Glu E Glutamine Gin Q Glycine Gly G Histidine His H Isoleucine lie I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V