The broad, long term goal of the proposed study is to determine, at the molecular level, the mechanisms by which DNA replication occurs in vivo. Faithful duplication of the genome is essential for life. Any disturbance in this process can lead to genomic instability, unregulated cell growth or cell death. Therefore, studying the mechanisms of DNA replication will contribute not only to broad areas of basic biology, but also to understanding the molecular basis for human diseases, such as cancer and developmental disorders. DNA replication requires highly coordinated recruitment of replication factors to replication origins in a chromatin context. It is therefore expected that histone modifications around replication origins strongly affect DNA replication. Accordingly, there is supporting evidence for significant roles of histone modifications in replication. However, how histone modifications are regulated around DNA replication origins and how these modifications affect different steps of DNA replication still remain as important open questions. In the current funding cycle of this grant, we have developed an extremely efficient system to purify histones that are located near an active replication origin. Combined with ultra-sensitive mass spectrometry analyses, we identified previously unknown patterns of histone modifications that are present specifically around an active replication origin. Furthermore, we have shown that these modifications play important roles in DNA replication and/or cellular responses to replication stress. In this funding cycle, we propose to extend these findings to elucidate how these newly identified histone modifications function at the molecular level.

Public Health Relevance

DNA replication and response to replication stress are conserved essential processes. Any abnormality in these processes can lead to cancer, developmental disorder or cell death. The goal of our work is to understand the molecular mechanisms of DNA replication and replication stress response, which will help understand the molecular basis for these disease states.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-GGG-N (02))
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Janes, Daniel E
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Fred Hutchinson Cancer Research Center
United States
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Rodriguez, Jairo; McKnight, Jeffrey N; Tsukiyama, Toshio (2014) Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM. Curr Protoc Mol Biol 108:21.28.1-16
Bogenschutz, Naomi L; Rodriguez, Jairo; Tsukiyama, Toshio (2014) Initiation of DNA replication from non-canonical sites on an origin-depleted chromosome. PLoS One 9:e114545
Rodriguez, Jairo; Tsukiyama, Toshio (2013) ATR-like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress. Genes Dev 27:74-86
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Unnikrishnan, Ashwin; Gafken, Philip R; Tsukiyama, Toshio (2010) Dynamic changes in histone acetylation regulate origins of DNA replication. Nat Struct Mol Biol 17:430-7
Ng, Wei DA; Wong, Chee Keong Benjamin (2007) SELF-RECOGNITION OF DNA FROM LIFE PROCESSES TO DNA COMPUTATION. Biophys Rev Lett 2:123-137