Accurate DNA replication and maintenance of genome integrity are essential for cell survival. DNA replication stress has been specifically associated with development of cancer. Malignancy is accompanied by progressive loss of genome integrity which can cause or exacerbate the disease. Dysregulation of the genes encoding the conserved MCM helicase occurs in cancer cells, and mcm mutations are also linked to cancer development in mice. These studies indicate that analysis of the mechanism by which the MCM complex contributes to genome stability have direct relevance to human health and disease. This proposal uses a tractable model system, the fission yeast S. pombe, to investigate the contributions of MCM proteins to chromosome integrity. It is known that mcm mutants cause replication fork collapse. This suggests that the MCM helicase is a crucial player in replication fork stability and chromosome dynamics during the cell cycle. The project uses genetics, molecular biology, and cell biology methods, including a new system for single-cell analysis to provide live cell imaging of the dynamics of stabilization and recovery.
The first aim analyzes replication fork components at normal, stalled and collapsed forks. It compares fork collapse in checkpoint mutants to that induced by mutation of the MCM helicase.
This aim examines the structure of the stalled or collapsed forks using molecular methods, and complements this by imaging fork proteins using a novel chromatin fiber technique.
In Aim 2, live cell analysis is used to examine the dynamics of fork recovery by assessing the recruitment of damage response and repair proteins to the damaged fork. It correlates this to events of the recovering cell cycle, and examines how cell division is disrupted if these responses are not normal. The live cell analysis is complemented by molecular approaches to examine repair and recovery proteins at individual replication forks.
Aim 3 takes a molecular approach to investigate evidence that the MCM complex, and particularly Mcm4, is a substrate of the replication checkpoint kinase Cds1. It analyzes mutant forms of Mcm4 that disrupt checkpoint response for their effects on protein recruitment and cell cycle dynamics, using the methods of Aim 1 and 2, and examines whether they affect association with the replication complex, checkpoint proteins, or repair proteins.
Aim 4 examines whether particular regions of the genome have increased sensitivity to MCM defects.
This aim uses genomic approaches to determine whether replication defects in mcm mutants cause stochastically distributed damage in the genome. Complementing the genomics studies, this aim concludes with the hypothesis that the ribosomal DNA may be particularly sensitive to mcm induced fork collapse, and investigates whether can be used as a model region for MCM-dependent genome integrity. Together, these aims provide a holistic picture of how a network of replication proteins interacts to maintain genome stability in the cell.

Public Health Relevance

Cancer results when cells suffer defects in DNA replication that affect chromosome stability. A network of conserved proteins normally functions to protect cells from DNA damage and replication defects, and mutations in these proteins are directly associated with cancer in humans. This project uses genetics and cell biology in a simple yeast to study how these conserved proteins normally work to protect the genome, and to study the molecular consequences when they are disrupted.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059321-12
Application #
8231430
Study Section
Nuclear Dynamics and Transport (NDT)
Program Officer
Reddy, Michael K
Project Start
1999-08-01
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2014-02-28
Support Year
12
Fiscal Year
2012
Total Cost
$457,775
Indirect Cost
$175,198
Name
University of Southern California
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Ding, Lin; Laor, Dana; Weisman, Ronit et al. (2014) Rapid regulation of nuclear proteins by rapamycin-induced translocation in fission yeast. Yeast 31:253-64
Ding, Lin; Forsburg, Susan L (2014) Essential domains of Schizosaccharomyces pombe Rad8 required for DNA damage response. G3 (Bethesda) 4:1373-84
Li, Pao-Chen; Green, Marc D; Forsburg, Susan L (2013) Mutations disrupting histone methylation have different effects on replication timing in S. pombe centromere. PLoS One 8:e61464
Forsburg, Susan L (2013) The CINs of the centromere. Biochem Soc Trans 41:1706-11
Li, Pao-Chen; Petreaca, Ruben C; Jensen, Amanda et al. (2013) Replication fork stability is essential for the maintenance of centromere integrity in the absence of heterochromatin. Cell Rep 3:638-45
Slaymaker, Ian M; Fu, Yang; Toso, Daniel B et al. (2013) Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology. Nucleic Acids Res 41:3446-56
Sabatinos, Sarah A; Mastro, Tara L; Green, Marc D et al. (2013) A mammalian-like DNA damage response of fission yeast to nucleoside analogs. Genetics 193:143-57
Li, Pao-Chen; Chretien, Louise; Cote, Jocelyn et al. (2011) S. pombe replication protein Cdc18 (Cdc6) interacts with Swi6 (HP1) heterochromatin protein: region specific effects and replication timing in the centromere. Cell Cycle 10:323-36
Ding, Lin; Forsburg, Susan L (2011) Schizosaccharomyces pombe minichromosome maintenance-binding protein (MCM-BP) antagonizes MCM helicase. J Biol Chem 286:32918-30
Dolan, William P; Le, Anh-Huy; Schmidt, Henning et al. (2010) Fission yeast Hsk1 (Cdc7) kinase is required after replication initiation for induced mutagenesis and proper response to DNA alkylation damage. Genetics 185:39-53

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