Identification Of Mde4,Swi5,Sfr1 Protein Complex By Tandem Affinity Purification And Mass Spectrometry

Accurate chromosome segregation during the cell division is crucial since cells containing an incomplete chromosome complement will either die or have abnormal growth phenotypes. Abundant evidence indicates that both kinetochore orientation and physical links between homologs, which are usually established through chromosome pairing, synapsis, and recombination, contribute to the faithful sister genomes segregation.Swi5 and Sfr1 are conserved eukaryotic proteins found in species ranging from yeast to human. Swi5 is involved in two related processes, the mating-type switching and homologous recombination. Genetic epistasis analysis suggested that there are two different Swi5- containing protein complexes in the fission yeast S. pombe. A protein complex containing Swi5 and Swi2 specifically promotes mating-type switching, whereas another complex containing Swi5 and Sfr1 is involved in Rhp51-dependent homologous recombinational repair. Rhp51 recombinase initiates a reciprocal DNA-strand exchange reaction between two homologous duplex DNA molecules by forming a helical nucleoprotein filament on single-stranded DNA. Accessory proteins reffered to as mediators are required for the full activity of the Rhp51 recombinase. Recent studies showed that Swi5-Sfr1 complex is a mediator that stabilizes and activates the Rhp51 filaments. On the other hand, Swi5-Swi2 complex is required for efficient mating-type switching which occurs during the cell cycle by gene conversion of the transcriptionally active mat1 locus using information from one of two silent cassettes. Mating-type switching is initiated by formation of the double strand break at the mat1 locus which recruits DNA repair machinery that facilitates the search for homologous donor sequences to repair the double strand break. Swi5 and Swi2 proteins exhibit cell type-specific localization pattern at the silent mating-type region. This differential localization modulates donor preference during mating-type switching. However, the molecular function of Swi5-Swi2 complex in this process remains unclear.The previous work of Gregan's lab has shown that Mde4, a monopolin protein, associated with Pcs1 is required for proper segregation of chromosome during mitosis and meiosis. Deletion of the mde4 gene, like that of pcs1, causes the appearance of lagging chromosomes during the anaphases of mitotic and meiosis II cells. They proposed that this monopolin complex can correct the merotelic attachment by clamping together multiple microtubule binding site on the same kinetochore.In the present experiment, we used fission yeast Schizosaccharomyces pombe as a model organism to study chromosome segregation. We have recently developed a new strategy to TAP-tag genes in the fission yeast. In this strategy, we amplified the region upstream and downstream of the stop codon of the target gene by PCR, These homology regions are 250-500 bp long. Restriction sites in the primers allow cloning of the homology regions into the pTAPKan1 vector carrying the TAP tag and the KanMX gene conferring resistance to geneticin (G418). The vector containing homology regions is amplified in E. coli and after linearization by a restriction enzyme transformed into yeast. Homologous recombination is used to replace the target gene with an allele encoding TAP-tagged version. Finally, successful tagging is verified in yeast transformants by colony PCR. We employed this strategy to tag S. pombe swi5, sfr1 and mde4 in-frame with a C-terminal TAP tag. Western blot analysis was conducted to prove the successful TAP tagging. After tandem affinity purification, we loaded the sample to SDS-PAGE gel, and the following silver staining can help to identify the best quality purified sample, then the proper purified proteins were subjected to analysis by mass- spectrometry.Results:1.We constructed one plasmid, namely pTAPKan1 vector, which carries the TAP tag, Amp and geneticin resistant gene. This new construction can be used to fuse the C-terminal TAP tag with target genes in the fission yeast S. pombe.2.We employed this TAP-tagging technology to fuse the gene of swi5, sfr1 and mde4 with C-terminal TAP tag followed by KanMX gene. Colony PCR has proved the successful tagging. While comparing with the traditional TAP-tagging strategy, which is usually based on PCR, our strategy has advantages of high transformation efficiency and specificity. 3.We purified the proteins tagged with TAP tag, and after analysis by mass spectrometry, we not only confirmed the already known Swi5-containing protein complexes, namely Swi5-Swi2 and Swi5-Sfr1, but also identified novel proteins co-purifying with Swi5-TAP and/or Sfr1-TAP. Notably, Swi5-TAP associated with Sir2 protein. A single unique Sir2 peptide was also identified in Sfr1-TAP purification. Members of the Sir2 family exist in species that range from prokaryotes to higher eukaryotes. Sir2 proteins have NAD(+)- dependent protein deacetylase activity and target for deacetylation histones H1, H3 and H4 as well as multiple non-histone proteins. Interestingly, Sir2 and Swi5 share similar biological functions. Both Sir2 and Swi5 are required for proper function of mating-type loci and efficient DNA repair. It is intriguing to speculate that the protein deacetylase activity of the Sir2 is required for the Swi5 function.4.We proved again by mass spectrometry, Mde4-TAP associated with chromosome segregation protein Pcs1, a subunit of the monopolin complex required for the orientation of sister kinetochores to the same pole (mono-orientation) during meiosis I, in addition, we also identify a novel protein, Dnt1, co-purifying with Mde4-TAP protein. It is reasonable to believe that Dnt1 may be involved in the process of accurate chromosome segregation.5.We found that both Swi5 and Sfr1 proteins were phosphorylated. Swi5 was phoshorylated on serine 72 and Sfr1 contained three phosphorylated serine residues (serins 26, 109 and 165). Further studies are required to analyze the functional relevance of Swi5 and Sfr1 phosphorylation and to identify the relevant protein kinases.