An important task of genome duplication is to overcome the large number of diverse obstacles posed by template lesions or DNA and protein blocks. Studies in the past decade suggest the existence of multiple mechanisms to rescue and complete replication in the face of these impediments;these include stabilization and remodeling of stalled replisomes to allow replication resumption, coordination of various types of DNA repair, and utilization of specialized DNA polymerases. Defects in these processes lead to debilitating human pathologies such as sterility, cancer, and developmental defects;thus studying these processes and understanding the underlying mechanisms, which are the goals of our studies, are of considerable significance to human health. Recent studies have shown that the evolutionarily conserved Smc5/6 complex is essential for growth and critical for promoting replication under normal and DNA damaging conditions. We have made significant progress in the first cycle of this grant in revealing the function and structure of this complex in the budding yeast model system. Results from our lab and others suggest that the Smc5/6 complex plays multiple roles in promoting replication;these include the prevention of accumulation of toxic recombination intermediates, the modification of replication and repair proteins with the small protein modifier SUMO, and collaboration with other replication regulatory factors. Currently how the Smc5/6 complex carries out these functions is not well understood, and research into these will shed light on the various replication-promoting processes. In the next grant period, we propose to elucidate the basis of the Smc5/6 complex function in replication using a combination of genetic, biochemical, and genomic approaches in the yeast model system.
In Aim 1, we will investigate how the Smc5/6 complex collaborates with another conserved replication regulatory factor Rtt107 to promote replication. We will use quantitative genome-wide measurements of DNA synthesis and replisome-DNA association to characterize the effects of these factors on replication. We will also examine the physical interactions between the Smc5/6 complex, Rtt107, and replication machinery.
In Aim 2, we will investigate how the Smc5/6 complex utilizes its SUMO ligase enzymatic activity to influence replication and repair. We have identified several key replication and repair enzymes whose sumoylation shows dependence on this complex. We will examine how the sumoylation of these substrates contributes to replication under different types of stress conditions.
In Aim 3, we will investigate the mechanisms that target the Smc5/6 complex and Rtt107 to stalled replication forks to enable replication regulation. These proposed studies have broad implications for understanding how replication is regulated to enable proper development and survival of all organisms.

Public Health Relevance

A network of proteins and multiple mechanisms collaborate to ensure successful replication;defects in these can lead to debilitating human pathologies, such as sterility, cancer and developmental defects. Our work of identifying proteins important for these processes and determining how they function will yield an understanding of replication regulation that contributes to faith genome duplication, thus are extremely important for human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM080670-06A1
Application #
8632499
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Reddy, Michael K
Project Start
2007-09-20
Project End
2017-12-31
Budget Start
2014-02-01
Budget End
2014-12-31
Support Year
6
Fiscal Year
2014
Total Cost
Indirect Cost
City
New York
State
NY
Country
United States
Zip Code
10065
Wei, Lei; Zhao, Xiaolan (2016) A new MCM modification cycle regulates DNA replication initiation. Nat Struct Mol Biol 23:209-16
Xue, Xiaoyu; Papusha, Alma; Choi, Koyi et al. (2016) Differential regulation of the anti-crossover and replication fork regression activities of Mph1 by Mte1. Genes Dev 30:687-99
Silva, Sonia; Altmannova, Veronika; Eckert-Boulet, Nadine et al. (2016) SUMOylation of Rad52-Rad59 synergistically change the outcome of mitotic recombination. DNA Repair (Amst) 42:11-25
Hang, Lisa; Zhao, Xiaolan (2016) The Rtt107 BRCT scaffold and its partner modification enzymes collaborate to promote replication. Nucleus 7:346-51
Wan, Bingbing; Hang, Lisa E; Zhao, Xiaolan (2016) Multi-BRCT scaffolds use distinct strategies to support genome maintenance. Cell Cycle 15:2561-2570
Bonner, Jaclyn N; Zhao, Xiaolan (2016) Replication-Associated Recombinational Repair: Lessons from Budding Yeast. Genes (Basel) 7:
Dummer, Antoinette M; Su, Zhangli; Cherney, Rachel et al. (2016) Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast. PLoS Genet 12:e1006094
Bonner, Jaclyn N; Choi, Koyi; Xue, Xiaoyu et al. (2016) Smc5/6 Mediated Sumoylation of the Sgs1-Top3-Rmi1 Complex Promotes Removal of Recombination Intermediates. Cell Rep 16:368-78
Choi, Koyi; Batke, Sabrina; Szakal, Barnabas et al. (2015) Concerted and differential actions of two enzymatic domains underlie Rad5 contributions to DNA damage tolerance. Nucleic Acids Res 43:2666-77
Xue, Xiaoyu; Choi, Koyi; Bonner, Jaclyn N et al. (2015) Selective modulation of the functions of a conserved DNA motor by a histone fold complex. Genes Dev 29:1000-5

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