Genomic instability is a major cause of various genetic disease, most notably cancer. To ensure genomic integrity, cell must replicate the millions or billions of DNA base pairs with absolute fidelity every cell cycle. However, cells are constantly under stress of many factors that cause DNA damage or block DNA replication. To circumvent these problems, cells are equipped with a quality control system, termed the DNA replication checkpoint. In humans, defects in this checkpoint cause genetic instability, leading to developmental and neurological defects, and a strong predisposition to cancer. Therefore, the long-term objective of this application is to elucidate how the checkpoints maintain genomic integrity to prevent a variety of genetic disorders. The Swi1-Swi3 complex, which is also known as the Replication Fork Protection Complex (FPC) , plays a central role in activation of the DNA replication checkpoint and stabilization of stalled replication forks in the fission yeast Schizosaccharomyces pombe. The Swi1-Swi3 complex is evolutionally conserved and homologous to Tof1-Csm3 in budding yeast Saccharomyces cerevisiae and Timeless-Tipin in humans. However, how the FPC stabilizes the replication forks, and how it controls the replication checkpoint, are unknown. Therefore, the first aim is to understand the molecular basis of the FPC at the replication forks. We will elucidate how the FPC recognizes the replication forks.
The second aims i s to establish the functional significance of Swi1 phosphorylation and DDK-FPC interaction. We will investigate the roles a Cdc7-like kinase complex in phosphorylation and regulation of the FPC.
The third aim i s to understand the roles of Mrc1-FPC interaction in fork stabilization and activation of the replication checkpoint. We will elucidate how the Mrc1 cooperates with FPC to regulate its function to understand the molecular mechanisms of fork stabilization and checkpoint signaling. These studies are designed to test the central hypothesis that FPC coordinates replication fork stabilization and checkpoint signaling through its association with replication forks and interaction with DDK and Mrc1. Genetic and biochemical studies will be carried out in the fission yeast Schizosaccharomyces pombe because it has been shown to be an exceptional model system for studying cell cycle control and genome maintenance mechanisms which are highly conserved amongst eukaryotes, including humans. Successful completion of these experiments should provide a much better framework for guiding investigations of genome maintenance mechanisms in humans. Project Narrative: Cells are constantly under the stress of many factors that arrest DNA replication. These factors often cause genetic alterations, which lead to developmental and neurological defects, and a variety of genetic diseases, most notably cancer. Therefore, elucidating how cells cope with replication arrest is essential for understanding the mechanisms of birth defects and development of cancer, contributing to the development of therapeutic agents. Project Narrative: Cells are constantly under the stress of many factors that arrest DNA replication. These factors often cause genetic alterations, which lead to developmental and neurological defects, and a variety of genetic diseases, most notably cancer. Therefore, elucidating how cells cope with replication arrest is essential for understanding the mechanisms of birth defects and development of cancer, contributing to the development of therapeutic agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077604-05
Application #
8231490
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Reddy, Michael K
Project Start
2008-05-01
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2014-02-28
Support Year
5
Fiscal Year
2012
Total Cost
$261,883
Indirect Cost
$85,465
Name
Drexel University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
002604817
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Kriete, Andres; Noguchi, Eishi; Sell, Christian (2014) Introductory review of computational cell cycle modeling. Methods Mol Biol 1170:267-75
Leman, Adam R; Noguchi, Eishi (2014) Chromatin immunoprecipitation to investigate origin association of replication factors in mammalian cells. Methods Mol Biol 1170:539-47
Leman, Adam R; Noguchi, Eishi (2014) Linking chromosome duplication and segregation via sister chromatid cohesion. Methods Mol Biol 1170:75-98
Noguchi, Eishi (2013) PP2A(Cdc55/B55), a possible therapeutic target in cyclin D1-dependent cancers. Cell Cycle 12:1484
Gadaleta, Mariana C; Iwasaki, Osamu; Noguchi, Chiaki et al. (2013) New vectors for epitope tagging and gene disruption in Schizosaccharomyces pombe. Biotechniques 55:257-63
Roseaulin, Laura C; Noguchi, Chiaki; Martinez, Esteban et al. (2013) Coordinated degradation of replisome components ensures genome stability upon replication stress in the absence of the replication fork protection complex. PLoS Genet 9:e1003213
Shah, Niyant; Inoue, Akira; Woo Lee, Seung et al. (2013) Roles of ChlR1 DNA helicase in replication recovery from DNA damage. Exp Cell Res 319:2244-53
Roseaulin, Laura C; Noguchi, Chiaki; Noguchi, Eishi (2013) Proteasome-dependent degradation of replisome components regulates faithful DNA replication. Cell Cycle 12:2564-9
Leman, Adam R; Noguchi, Eishi (2013) The replication fork: understanding the eukaryotic replication machinery and the challenges to genome duplication. Genes (Basel) 4:1-32
Garabedian, Mikael V; Noguchi, Chiaki; Ziegler, Melissa A et al. (2012) The double-bromodomain proteins Bdf1 and Bdf2 modulate chromatin structure to regulate S-phase stress response in Schizosaccharomyces pombe. Genetics 190:487-500

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