The faithful propagation of chromosomes is crucial to suppress aneuploidy-related birth defects, and cancer. Therefore, our overarching objective is to define key mechanisms within the DNA repair, replication and cell cycle pathways that support accurate chromosome transmission. This proposal centers on the evolutionarily conserved Smc5-Smc6 holocomplex and its cofactor, Rad60;which are master regulators of genome stability and DNA repair. We identified the eight core subunits of the Smc5-Smc6 complex, revealing that unlike the related cohesin and condensin complexes, Smc5-Smc6 can regulate the action of other proteins by modifying them with SUMO and/or ubiquitin. In addition, we discovered that Rad60 mimics SUMO by forming a structurally analogous non-covalent complex with Ubc9, and in this way facilitates Smc5-Smc6-mediated SUMOylation. This discovery explains the observed functional overlap between Smc5-Smc6, Rad60 and SUMO in genome maintenance. Furthermore, we have recently revealed critical but mechanistically undefined functions for Smc5-Smc6 in (i) promoting meiotic chromosome segregation at the MI division, (ii) the processing of protein-DNA adducts in a pathway parallel to Tdp1, and (iii) the resolution of DNA structures arising during the restart of collapsed replication forks through homologous recombination-based repair. Building on this foundation and compelling preliminary data, we propose to elucidate the mechanisms of Smc5- Smc6 and Rad60 in these specific chromosome segregation and DNA repair processes. We will achieve our goal by integrating genetics, biochemistry and mass spectrometry experiments in the proven fission yeast model organism. We have two Specific Aims.
Aim 1 : To define the mechanism(s) of Smc5-Smc6 in processing Holliday junctions, which are homologous recombination-dependent covalent chromosome linkages generated during meiosis and replication fork restart. Also, the pathways and proteins through which Smc5-Smc6 and Rad60 promote the repair of genotoxic protein-DNA adducts (e.g. Top1cc) will be determined.
Aim 2 : To define the Smc5-Smc6 genome stabilizing """"""""network"""""""" through: (i) characterization of novel dosage suppressors of hypomorphic Smc5-Smc6 subunits, and (ii) testing the physiological impact of Smc5-Smc6-mediated sumoylation on target proteins that we recently identified. Although each of our Aims is self-standing, the results from each will likely synergize to provide rapid insight into the critical functions of Smc5-Smc6 and Rad60. Overall, completion of our Aims will significantly deepen our understanding of specific genome stability mechanisms, which are relevant to both the etiology and treatment of human disease.
It is imperative that we define and understand the detailed workings of the cellular machinery that ensures accurate passage of chromosomes;as when defective, changes are made to our program that can manifest in a number of ways, including birth defects and cancer. Exploiting the conservation of these critical chromosome guardians throughout evolution;we propose to study the key factors Smc5-Smc6 and Rad60 in the fission yeast model organism, which is proven to provide rapid insight on disease relevant processes. Through our proposed studies we expect to generate pivotal knowledge on the etiology of birth defects and cancer, and characterize novel targets for improved cancer therapies.
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