The eukaryotic ATP-ases of SMC (structural maintenance of chromosomes) family are in the core of two essential eukaryotic protein complexes: cohesin and condensin, which determine high order chromosome structure in proliferating cells. To understand the molecular mechanisms of these SMC-containing complexes we combine genetic, cytological and biochemical approaches. The focus of condensin studies was on the mechanisms determining its targeting specificity to the defined chromatin sites in mitosis. We screened the collection of mutants in chromatin proteins and cell-cycle mutants for defects in condensin targeting to rDNA in mitosis. We introduced the construct expressing fusion of condensin component Smc4p to GFP into each strain. Several candidate mutants were identified, that failed to re-localize the GFP-labelled condensin to rDNA in mitosis-specific manner. One of these mutants, cdc14-1 disrupts the mitosis exit network. The inability of condensin to re-localize to rDNA in mitosis in this mutant suggests that the Cdc14p phosphatase may play a direct role in regulation of condensin subunits. A deletion mutant of SMT4, encoding the Smt3p isopeptidase, was also among the mutants identified in the screen. We characterized the Smt4p protein and established that loss of Smt4p function induces chromosome instability, checkpoint defects and change in Smt3p-modification pattern of chromatin proteins. To find the essential targets of Smt3p conjugation among chromatin proteins we investigated the link between the Smt3p-modification and mitosis-specific condensin targeting to rDNA. In the course of these studies we isolated two genes, SIZ1 and SIZ2, which bypass the requirement for Smt4p when over-expressed. Siz1p is itself a chromatin component and largely co-localizes with condensin in vivo. In addition, loss of SIZ1, SIZ2 or SIZ1 and SIZ2 function in the corresponding deletion mutants changes selectivity of the Smt3p-conjugation machinery, suggesting that SIZ1 and SIZ2 may represent an elusive E3-like component of the Smt3p-conjugation pathway. Sister chromatid cohesion includes establishment and maintenance of physical association between sister chromatids by the cohesin complex from the S-phase till anaphase. To study the architecture of mitotic cohesin we purified individual recombinant cohesin subunits from insect cells and assembled cohesin complex using co-infection with recombinant baculoviruses. We also purified the ATP-ase core of cohesin: a heterodimer between the recombinant Smc1p and Smc3p proteins. We found that DNA-binding properties of cohesin, Smc1p/Smc3p dimer, and non-SMC subunits Scc3p and Mcd1p are very similar. All proteins display preference for AT-rich DNA but are unable to make a distinction in vitro between the DNA probes corresponding to known chromosomal cohesion sites and DNA from to the sites with no cohesion in vivo. The experiments with the defined chromatinised probe established that both Smc1p/Smc3p dimer and full cohesin bind chromatin with a very high affinity, while Mcd1p and Scc3p do not. The mode of Smc1p/Smc3p binding to chromatin was distinct from the full cohesin complex, which showed evidence of linking several chromatinised probes in a reaction potentially reproducing sister chromatid cohesion process in vitro.
NIH ExPorter
NIH ExPorter