THE ROLE OF SMC-FAMILY
PROTEINS AND ASSOCIATED FACTORS IN CHROMOSOME SEGREGATION
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Alexander
V. Strunnikov, Ph.D., Principal Investigator Vladimir Yong-Gonzales, Ph.D., Postdoctoral Fellow Alexander Kagansky, Graduate Student and Professional Services Contractor Abram Geisendorfer, Predoctoral Fellow Dmitry Lukyanov, Predoctoral Fellow |
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The family of eukaryotic SMC (structural maintenance of chromosomes) ATPases and associated subunits form two types of complex in eukaryotic organisms. These protein complexes, termed cohesin and condensin, largely determine high-order chromosome structure in proliferating cells. The Unit on Chromosome Structure and Function is using a combination of genetic, cytological, and biochemical approaches to elucidate the molecular mechanisms and detailed biological functions of these SMC-containing complexes. Analysis of Mitotic Chromosome Condensation and Controlling Mechanisms
Ensuring Proper Targeting of Condensin Complex to Chromatin The main focus of our studies on condensin was the mechanisms determining condensin's binding (targeting) specificity to the specific and recurring chromatin sites in mitosis. We applied cell biology and genetic approaches to screen the collection of mutants in chromatin proteins and cell-cycle mutants for defects in condensin targeting to rDNA in mitosis. Each mutant strain was transformed with the integrative plasmid expressing SMC4-GFP. As a result of screening for defects in mitosis-specific relocalization of GFP signal to rDNA, we identified several candidate mutants and investigated a possible direct role in condensin regulation. One of these mutants, cdc14-1, disrupts the mitosis exit network (MEN). The inability of condensin to relocalize to rDNA in mitosis in cdc14-1 suggests that the Cdc14p phosphatase may play a direct role in regulation of condensin subunits. The detected physical interaction of Cdc14p with condensin further supports our hypothesis. A deletion mutant in the Smt3p(SUMO) hydrolase-encoding gene SMT4 was also among the mutants identified in this screen. We characterized the Smt4p protein and established that it plays a pleiotropic role in chromosome maintenance. Loss of Smt4p function induces chromosome instability, checkpoint defects, and a change in the Smt3p-modification pattern of chromatin proteins. It is currently unknown which chromatin components depend on sumolation for their function. Yet, the gene encoding Smt3p in yeast (SMT3) is essential for cell viability. Thus, finding the essential Smt3 conjugation targets among chromatin proteins should reveal some important properties of chromatin organization. We conducted several experiments to elucidate the link between Smt3p-modification and mitosis-specific condensin targeting to rDNA. In the course of our studies, we isolated two genes, SIZ1 and SIZ2, that bypass the requirement for Smt4p when overexpressed. SIZ1p is itself a chromatin component and largely colocalizes with condensin in vivo. In addition, loss of SIZ1, SIZ2, or SIZ1 and SIZ2 function in the corresponding deletion mutants changes the selectivity of the Smt3p-conjugation machinery, resulting in specific changes in the array of modified targets. This finding suggests that SIZ1 and SIZ2 may be a long-sought E3 component of the Smt3p-conjugation pathway. We are continuing to investigate the roles of SIZ1 and SIZ2in chromosome condensation regulation. Analysis of Sister Chromatid Cohesion in S. cerevisiae
To characterize the biochemical properties of yeast cohesin, we raised polyclonal antibodies against all cohesin subunits and known auxiliary proteins. Purification from yeast cells did not, however, yield enough material for the in vitro assays because of the low abundance and tight chromatin association of cohesin. To assess the architecture of mitotic cohesin and putative meiotic cohesin, we purified recombinant Smc1p, Smc3p, Scc3p, Mcd1p, and Rec8p from insect cells. We established that the fifth protein, Pds5p, whose association with cohesin was reported, is bound to cohesin only transiently in vivo and is not a part of a biochemically defined cohesin complex. In addition, we were able to assemble cohesin in insect cells by using coinfection with four recombinant baculoviruses. We also purified a heterodimer between the recombinant Smc1p and Smc3p. Our current hypothesis is that the SMC components of cohesin, Smc1p, and Smc3p, are constitutive chromatin components that act as a molecular motor using ATP hydrolysis to glue together chromatin of sister chromatids after Mcd1p and Scc1p are expressed in early S phase. Mcd1p and Scc1p are thus absolutely required to establish and maintain links between chromatids. This hypothesis predicts that the Smc1p/Smc3p and the full four-subunit cohesin should have markedly different DNA- and chromatin-binding properties. Surprisingly, we found that the DNA-binding properties of cohesin, Smc1p/Smc3p dimer, Scc3p, and Mcd1p are very similar. All proteins display preference for AT-rich DANN, and the binding constant is very low. In addition, none of the tested proteins was able to make a distinction between the DNA probes corresponding to known in vivo cohesion sites and DNA corresponding to the sites with no cohesion in vivo. We conducted similar experiments with the defined chromatinised probe and established that both Smc1p/Smc3p dimer and full cohesin bind to chromatin with a very high affinity. The mode of Smc1p/Smc3p and cohesin binding to chromatin was clearly distinct: the full cohesin complex showed evidence of linking several chromatinised probes in a reaction that may potentially represent reconstitution of SCC in vitro. Currently, we are in the process of mapping the chromatin-binding site in cohesin complex and establishing which nucleosomal domains are responsible for cohesin binding. |
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