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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM068608-10A1
Application #
8503325
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Janes, Daniel E
Project Start
2003-07-01
Project End
2017-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
10
Fiscal Year
2013
Total Cost
$376,631
Indirect Cost
$177,881
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Nie, Minghua; Boddy, Michael N (2016) Cooperativity of the SUMO and Ubiquitin Pathways in Genome Stability. Biomolecules 6:14
Nie, Minghua; Arner, Emily; Prudden, John et al. (2016) Functional Crosstalk between the PP2A and SUMO Pathways Revealed by Analysis of STUbL Suppressor, razor 1-1. PLoS Genet 12:e1006165
Nie, Minghua; Boddy, Michael N (2015) Pli1(PIAS1) SUMO ligase protected by the nuclear pore-associated SUMO protease Ulp1SENP1/2. J Biol Chem 290:22678-85
Nie, Minghua; Vashisht, Ajay A; Wohlschlegel, James A et al. (2015) High Confidence Fission Yeast SUMO Conjugates Identified by Tandem Denaturing Affinity Purification. Sci Rep 5:14389
Groocock, Lynda M; Nie, Minghua; Prudden, John et al. (2014) RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response. EMBO Rep 15:601-8
Zilio, Nicola; Codlin, Sandra; Vashisht, Ajay A et al. (2014) A novel histone deacetylase complex in the control of transcription and genome stability. Mol Cell Biol 34:3500-14
Wehrkamp-Richter, Sophie; Hyppa, Randy W; Prudden, John et al. (2012) Meiotic DNA joint molecule resolution depends on Nse5-Nse6 of the Smc5-Smc6 holocomplex. Nucleic Acids Res 40:9633-46
Zilio, Nicola; Wehrkamp-Richter, Sophie; Boddy, Michael Nicholas (2012) A new versatile system for rapid control of gene expression in the fission yeast Schizosaccharomyces pombe. Yeast 29:425-34
Groocock, Lynda M; Prudden, John; Perry, J Jefferson P et al. (2012) The RecQ4 orthologue Hrq1 is critical for DNA interstrand cross-link repair and genome stability in fission yeast. Mol Cell Biol 32:276-87
Nie, Minghua; Aslanian, Aaron; Prudden, John et al. (2012) Dual recruitment of Cdc48 (p97)-Ufd1-Npl4 ubiquitin-selective segregase by small ubiquitin-like modifier protein (SUMO) and ubiquitin in SUMO-targeted ubiquitin ligase-mediated genome stability functions. J Biol Chem 287:29610-9

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