Smc (structural maintenance of chromosomes) complexes mediate higher-order chromosome structure by tethering together different regions of chromosomes. One Smc complex, cohesin, functions in several DNA processes, including those that require tethers between chromosomes (sister chromatid cohesion and DNA repair) and those that require tethers between two regions in the same chromosome (condensation and transcription regulation). These diverse functions of cohesin are thought to regulate gene expression in stem cells and to prevent errors in chromosome transmission that lead to cancer and birth defects. Cohesin has a remarkable ring architecture resulting from the interactions of four its subunits, Smc1, Smc3, Scc3, and Mcd1 (also known as Scc1/Rad21) (Fig. 1). Cohesin also contains two ATPases associated with the Smc subunits. These remarkable biological and biochemical properties of cohesin inspire the two questions that drive the aims in this proposal: 1) How do cohesin's architecture and ATPase activities mediate its binding to a DNA duplex and enable it to tether two different DNA duplexes? and 2) How is cohesin regulated to ensure that it functions properly in very diverse biological processes? Aim 1 explores the critical but unexplored asymmetric role(s) of the Smc3 and Smc1 ATPases in regulating cohesin binding to chromosomes and chromosome tethering in vivo.
Aim 2 characterizes a newly-detected cohesin activity, its oligomerization and its potential to generate tethers. Experiments in this aim will: ) test the role of cohesin oligomerization in cohesion and condensation; 2) interrogate how oligomerization changes during cell cycle progression, is modulated by DNA binding and controlled by cohesin regulators; and 3) identify the amino acid sequences within the Smc subunits that mediate oligomerization. By studying cohesin through the novel roles of the Smc ATPases and cohesin oligomerization, Aims 1 and 2 will elucidate unexplored aspects of cohesin function in vivo.
In Aim 3, the molecular basis for cohesin's in vivo activities will be interrogated by in vitro assays to measure cohesin's binding and diffusion along DNA, its oligomerization, its tethering activities, and any structural changes associated with these activities. These analyses will provide the framework to develop a specific molecular model for cohesin's DNA binding and tethering activities.
Aim 4 will explore how a newly identified group of regulators parse cohesin function prior to anaphase to ensure the proper maintenance of sister chromatid cohesion and the establishment of condensation. These analyses will provide insights into cohesin's essential functions in chromosome segregation and provide a paradigm for how the activities of Smc complexes are regulated to carry out distinct biological functions. The four aims exploit unusual genetic alleles that trap distinct functional states of cohesin, new cell biological, genetic and biochemical assays for cohesin oligomerization and new population and single molecule assays for assessing cohesin DNA binding and tethering in vitro.
Chromosomes are composed of very long threads of DNA that are packaged by proteins. Protein complexes like cohesin act as tethers to crosslink together different chromosomes or different regions within a chromosome. The crosslinks generated by cohesin are important for ensuring the proper inheritance of chromosomes during cell division and gamete formation, the repair of DNA damage and the proper expression of genes including those in stem cells. Defects in cohesin have been implicated in birth defects and cancer. This grant proposes to interrogate how cohesin crosslinks DNA, and how the cell regulates cohesin to carry out its remarkable biological and medically relevant functions.
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