The faithful transmission of undamaged chromosomes to daughter cells at cell division is essential for life and for suppression of human disease. Genomic DNA is packaged into proteinaceous chromatin, upon which the crucial processes of transcription, DNA replication and repair occur. These processes are facilitated by the evolutionarily conserved SMC family, which includes cohesin (Smc1-Smc3), required for sister chromatid cohesion and centromere function;condensin (Smc2-Smc4), required with topoisomerase II to compact and drive the decatenation of chromosomes at mitosis;and the Smc5-Smc6 complex, whose essential function(s) are undefined. Our long-term objective is to dissect and define critical regulatory interfaces between the DNA repair, replication and cell cycle progression mechanisms. Ultimately, this will yield a molecular level understanding of the interplay between these processes that should explain the etiology of many human diseases and perhaps highlight potential therapeutic strategies. Currently, we are defining the functional interface between the Smc5-Smc6 complex and the replication monitoring checkpoint, enforced by Cds1 (hCHK2). Our studies utilize the genetically tractable and well-proven fission yeast model organism. For our Specific Aims below, fission yeast is ideal, as it has complex heterochromatic centromeres and associated factors that are well conserved in humans. Furthermore, components of the replication checkpoint are also highly conserved. We will characterize the Smc5-Smc6 holocomplex by integrating powerful yeast genetics with cutting-edge structural experiments, biochemistry and mass spectrometry. We have two Specific Aims in which we will determine the mitotic and meiotic chromosome segregation role(s) of Smc5-Smc6 and the associated DNA repair protein, Rad60. As Rad60 physically interacts with both Smc5-Smc6 and the replication checkpoint kinase Cds1, it provides an interface between the replication checkpoint and DNA repair processes. In our first Aim we will test the physiological importance of the Cds1-Rad60 interface in suppressing toxic replication-associated recombination. In addition, Rad60 family proteins contain the unique structural signature of tandem SUMO-like domains (SLDs). Therefore, we will functionally characterize Rad60 by solving the structures of its SLDs, which will facilitate mutagenesis coupled with in vivo phenotypic analyses. These mutagenesis studies will define the mechanism(s) by which Rad60 mediates its observed role in heterochromatin formation and stability. In our second Aim we will characterize the mitotic and meiotic homologous recombination repair and centromeric functions of the Smc5-6 complex. This will be greatly facilitated by identifying targets of the SUMO E3 ligase Smc5-Smc6 subunit Nse2. These studies will yield a detailed picture of the ways in which the evolutionarily conserved Smc5-Smc6, Rad60 and replication checkpoint maintain chromosome integrity and thus in humans, suppress disease priming genomic lesions.

Public Health Relevance

We have identified a molecular """"""""machine"""""""" that protects and repairs the cells """"""""blueprint"""""""" or genome. The genome contains information required to maintain normal cell growth and if this information is damaged, cancer can ensue. Our studies will provide key insights on normal regulation of cell growth and furthermore, reveal a potential """"""""Achilles'Heel"""""""" of cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068608-09
Application #
8084149
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Janes, Daniel E
Project Start
2003-07-01
Project End
2013-03-31
Budget Start
2011-07-01
Budget End
2013-03-31
Support Year
9
Fiscal Year
2011
Total Cost
$371,458
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Nie, Minghua; Moser, Bettina A; Nakamura, Toru M et al. (2017) SUMO-targeted ubiquitin ligase activity can either suppress or promote genome instability, depending on the nature of the DNA lesion. PLoS Genet 13:e1006776
Zilio, Nicola; Boddy, Michael N (2017) Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes from Schizosaccharomyces pombe. Cold Spring Harb Protoc 2017:pdb.prot091611
Nie, Minghua; Boddy, Michael N (2017) Large-Scale Purification of Small Ubiquitin-Like Modifier (SUMO)-Modified Proteins from Schizosaccharomyces pombe. Cold Spring Harb Protoc 2017:pdb.prot091603
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; 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
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
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
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
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

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