The long-term goal of the proposed research is to understand how DNA replication is regulated such that a precise copy of the genome is made. Accurate replication of the genome and continuous surveillance of its integrity are essential for cell survival and the avoidance of diseases such as cancer and premature aging. The genome is constantly exposed to environmental and endogenous genotoxic insults that challenge DNA replication. The replication stress response (RSR) is a subset of the DNA damage response (DDR) that acts during every cell division cycle to deal with these challenges and promote the faithful duplication of the genome. One component of the replication stress response pathway is the checkpoint kinase ATR. ATR promotes faithful DNA replication by regulating origin firing and cell cycle progression, stabilizing stalled replication forks, and promoting the repair and recovery of stalled forks. The mechanisms mediating many of these ATR-regulated activities are poorly understood. We have completed a series of genetic and biochemical screens to define the mechanisms by which the replication stress response regulates DNA replication and maintains genetic stability. These screens included two-hybrid and mass-spectrometry screens to identify proteins that interact with the ATR-ATRIP complex and the TopBP1 regulator of ATR;RNAi screens to define genes that are important for cells to recover from replication fork stress;and RNAi and cDNA overexpression screens to identify genes that when deregulated cause genetic instability. By focusing on proteins identified in more than one of these biochemical and genetic screens and using published genomic/proteomic information, we identified thirty-six high confidence RSR proteins. These include twenty-four candidate RSR proteins not previously linked to replication stress responses. We hypothesize that these novel RSR proteins have distinct functions in replication and DNA damage responses. This hypothesis will be tested using a combination of genetic and biochemical approaches. Completion of this research proposal will define the genome maintenance activities of novel replication stress response proteins and improve our understanding of how a precise copy of the genome is made during every cell division cycle.

Public Health Relevance

Accurate replication of the genome and continuous surveillance of its integrity are essential for cell survival and the avoidance of diseases such as cancer and premature aging. The genome is constantly exposed to environmental and endogenous genotoxic insults that challenge DNA replication. This research proposal aims to define the genome maintenance functions of novel replication stress response proteins.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA136933-05
Application #
8512561
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Pelroy, Richard
Project Start
2009-09-21
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
5
Fiscal Year
2013
Total Cost
$293,258
Indirect Cost
$104,059
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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Carroll, Clinton; Bansbach, Carol E; Zhao, Runxiang et al. (2014) Phosphorylation of a C-terminal auto-inhibitory domain increases SMARCAL1 activity. Nucleic Acids Res 42:918-25
Couch, Frank B; Cortez, David (2014) Fork reversal, too much of a good thing. Cell Cycle 13:1049-50
Sirbu, Bianca M; McDonald, W Hayes; Dungrawala, Huzefa et al. (2013) Identification of proteins at active, stalled, and collapsed replication forks using isolation of proteins on nascent DNA (iPOND) coupled with mass spectrometry. J Biol Chem 288:31458-67
Betous, Remy; Couch, Frank B; Mason, Aaron C et al. (2013) Substrate-selective repair and restart of replication forks by DNA translocases. Cell Rep 3:1958-69
Nagarajan, Prabakaran; Ge, Zhongqi; Sirbu, Bianca et al. (2013) Histone acetyl transferase 1 is essential for mammalian development, genome stability, and the processing of newly synthesized histones H3 and H4. PLoS Genet 9:e1003518
Couch, Frank B; Bansbach, Carol E; Driscoll, Robert et al. (2013) ATR phosphorylates SMARCAL1 to prevent replication fork collapse. Genes Dev 27:1610-23
Betous, Remy; Glick, Gloria G; Zhao, Runxiang et al. (2013) Identification and characterization of SMARCAL1 protein complexes. PLoS One 8:e63149
Sirbu, Bianca M; Cortez, David (2013) DNA damage response: three levels of DNA repair regulation. Cold Spring Harb Perspect Biol 5:a012724
Sirbu, Bianca M; Couch, Frank B; Cortez, David (2012) Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA. Nat Protoc 7:594-605

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