DNA replication is a fundamental process essential for growth and development. Errors that occur during DNA replication have the potential to lead to genome instability resulting in various diseases, including cancer. DNA replication is intrinsically coupled with the developmental program of organisms; miss- regulation of DNA replication can lead to disabling syndromes. Although DNA replication has been actively studied for decades there are numerous aspects that are still poorly understood. Foremost is the fundamental question of what specifies the location of replication origins in metazoan cells? A body of data points to the general association of replication origins with sites of active gene transcription, but the underlying factors that specify origins remain unknown. The profound cellular changes that occur during embryonic development offer an excellent system to study how gene transcription and DNA replication programs are established and maintained. An accurate map of how DNA replication, transcription and histone modification patterns change through embryogenesis will provide a simple, yet powerful means to define how such processes are spatially and temporally related. My lab has developed and optimized a method to map DNA replication, which involves the purification and sequencing of Okazaki fragments. This has proven to be a very robust assay, in this proposal we will utilize Okazaki fragment sequencing to define how origins in metazoa are specified. We have developed C. elegans as a model system to study DNA replication and have generated a large body of data that highlights the utility this organism. Our data shows that replication initiates from gene enhancers, and are defined by histone modifications. Thus, our preliminary data reveals a novel link between chromatin structure, transcription and replication origins. In this proposal, we will use a range of genomic, and molecular biological approaches. We will take advantage of the profound changes that occur during embryonic development to test whether gene transcription or histone modifications are required to establish specific replication origins; in addition, we examine whether non-coding RNA at replication origins plays a role in origin function. Finally we explore whether gene enhancers function to promote both gene transcription and DNA replication. Our interdisciplinary work will address key problems that were previously intractable. Our studies will be directly relevant to many research programs and will translate into a better understanding of the biological processes fundamental to maintaining genomic integrity and chromatin states.

Public Health Relevance

Each time a cell divides it must produce an accurate copy of its genome. This process, termed DNA replication, is essential and defects in DNA replication can lead to mutations, chromosomal rearrangements and cancer. This project will use new technology to understand the molecular mechanisms that control DNA replication.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Reddy, Michael K
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Sloan-Kettering Institute for Cancer Research
New York
United States
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Bellush, James M; Whitehouse, Iestyn (2017) DNA replication through a chromatin environment. Philos Trans R Soc Lond B Biol Sci 372:
Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya et al. (2017) Chromatin Constrains the Initiation and Elongation of DNA Replication. Mol Cell 65:131-141
Mattiroli, Francesca; Gu, Yajie; Yadav, Tejas et al. (2017) DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication. Elife 6:
Pourkarimi, Ehsan; Bellush, James M; Whitehouse, Iestyn (2016) Spatiotemporal coupling and decoupling of gene transcription with DNA replication origins during embryogenesis in C. elegans. Elife 5:
Yadav, Tejas; Whitehouse, Iestyn (2016) Replication-Coupled Nucleosome Assembly and Positioning by ATP-Dependent Chromatin-Remodeling Enzymes. Cell Rep 15:715-723
Smith, Duncan J; Yadav, Tejas; Whitehouse, Iestyn (2015) Detection and Sequencing of Okazaki Fragments in S. cerevisiae. Methods Mol Biol 1300:141-53
Gros, Julien; Kumar, Charanya; Lynch, Gerard et al. (2015) Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA. Mol Cell 60:797-807
McGuffee, Sean R; Smith, Duncan J; Whitehouse, Iestyn (2013) Quantitative, genome-wide analysis of eukaryotic replication initiation and termination. Mol Cell 50:123-35
Borges, Vanessa; Smith, Duncan J; Whitehouse, Iestyn et al. (2013) An Eco1-independent sister chromatid cohesion establishment pathway in S. cerevisiae. Chromosoma 122:121-34
Whitehouse, Iestyn; Smith, Duncan J (2013) Chromatin dynamics at the replication fork: there's more to life than histones. Curr Opin Genet Dev 23:140-6

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