DNA replication must be regulated precisely to maintain gene copy number. Cancer cells contain many regions in which gene copy number has been amplified and these are associated with tumor progression, but the primary mechanisms causing gene amplification are unknown. Failure of replication may contribute to chromosome fragile sites that lead to chromosomal instability. Mutations in proteins needed for replication initiation are associated with human developmental defects. It is crucial to determine how replication origins are activated or inactivated and how replication fork progression is controlled in metazoan cells. This necessitates the analysis of individual origins, the localization of replication initiation proteins, and ways to examine fork progression. This research will exploit developmental models of genomic regions that become amplified in copy number or under-replicated in response to differentiation in Drosophila. These models provide defined replication origins to which replication initiation proteins, essential for replication in human cells, can be localized and that can be analyzed in differentiated cells as they undergo initiation of DNA replication. They also define specific domains through which replication fork progression is permitted or impeded. These models provide a paradigm for delineating the mechanisms of replication initiation and elongation not present in another metazoan system.
The Specific Aims of this proposal are to use these models to answer fundamental questions about replication initiation and fork progression, utilizing interdisciplinary approaches of genomics, genetics and biochemistry. The mechanisms leading to origin activation will be defined for two amplified domains, regions of the genome permissive or restrictive for initiation will be investigated to determine how the Origin Recognition Complex (ORC) is localized and whether there are ORC- independent mechanisms of initiation. In the second aim, genomic regions that block initiation will be used to delineate the effects of chromatin configuration on ORC binding and origin activation. The last aim is to determine how replication fork progression is controlled by chromatin by identifying proteins that promote or block fork movement. Candidate proteins identified by mutants and localization patterns will be investigated.
This aim also will analyze th requirement for double-strand break repair at the forks.

Public Health Relevance

Proper regulation of DNA replication is essential to prevent changes in gene copy number that are prevalent in cancer cells and associated with tumor progression. Defects in DNA replication also may contribute to chromosome fragile sites associated with mental retardation. We have developed models for metazoan DNA replication and will exploit these to delineate the control mechanisms that activate or repress DNA replication.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057960-15
Application #
8665432
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Reddy, Michael K
Project Start
1999-05-01
Project End
2016-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
15
Fiscal Year
2014
Total Cost
$399,750
Indirect Cost
$194,750
Name
Whitehead Institute for Biomedical Research
Department
Type
DUNS #
120989983
City
Cambridge
State
MA
Country
United States
Zip Code
02142
Nordman, Jared T; Kozhevnikova, Elena N; Verrijzer, C Peter et al. (2014) DNA copy-number control through inhibition of replication fork progression. Cell Rep 9:841-9
Sher, Noa; Bell, George W; Li, Sharon et al. (2012) Developmental control of gene copy number by repression of replication initiation and fork progression. Genome Res 22:64-75
Kim, Jane C; Nordman, Jared; Xie, Fang et al. (2011) Integrative analysis of gene amplification in Drosophila follicle cells: parameters of origin activation and repression. Genes Dev 25:1384-98
Kim, Jane C; Orr-Weaver, Terry L (2011) Analysis of a Drosophila amplicon in follicle cells highlights the diversity of metazoan replication origins. Proc Natl Acad Sci U S A 108:16681-6
Nordman, Jared; Li, Sharon; Eng, Thomas et al. (2011) Developmental control of the DNA replication and transcription programs. Genome Res 21:175-81
Park, Eugenia A; Macalpine, David M; Orr-Weaver, Terry L (2007) Drosophila follicle cell amplicons as models for metazoan DNA replication: a cyclinE mutant exhibits increased replication fork elongation. Proc Natl Acad Sci U S A 104:16739-46
Lee, Laura A; Orr-Weaver, Terry L (2003) Regulation of cell cycles in Drosophila development: intrinsic and extrinsic cues. Annu Rev Genet 37:545-78
Claycomb, Julie M; MacAlpine, David M; Evans, James G et al. (2002) Visualization of replication initiation and elongation in Drosophila. J Cell Biol 159:225-36
Kashevsky, Helena; Wallace, Julie A; Reed, Bruce H et al. (2002) The anaphase promoting complex/cyclosome is required during development for modified cell cycles. Proc Natl Acad Sci U S A 99:11217-22
Royzman, Irena; Hayashi-Hagihara, Aki; Dej, Kimberley J et al. (2002) The E2F cell cycle regulator is required for Drosophila nurse cell DNA replication and apoptosis. Mech Dev 119:225-37

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