The overall goal of this project is to elucidate the molecular mechanisms that regulate the activity of DNA replication origins in S. cerevisiae. The origin recognition complex (ORC) directly binds to and regulates the function of eukaryotic replication origins by participating in the assembly of replication complexes that duplicate the chromosomal DNA. Assembly and activation of origin complexes is regulated by the cell cycle and checkpoints to ensure the accuracy of chromosome duplication prior to mitotic segregation. Chromatin structure also regulates origin function through unknown mechanisms. Our guiding hypothesis is that regulation of the interactions of ORC with replication origins is a critical determinant of subsequent origin activity. Using cellular, biochemical, and genetic analyses, we will characterize the cell cycle interactions of ORC with replication origins in S. cerevisiae, focussing on how the chromatin environment influences ORC-origin interactions and origin function.
The specific aims of this research arc to: 1) determine how the characteristics of ORC-origin binding regulate the activity of each origin, 2) elucidate the relationship between DNA replication and chromatin structure by analyzing the structural modifications of histones at differently regulated origins, and 3) characterize these interactions throughout the genome. ? ? Strict regulation of the activity of replication origins ensures accuracy in the transmission of the genome to daughter cells, proper regulation of cellular proliferation, and integration of cell duplication with gene expression and cellular differentiation. Understanding how replication origin activity is regulated in all of its contexts has the potential of providing improved methods for the detection and treatment of cancer, as well as elucidating mechanisms of development.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cell Development and Function Integrated Review Group (CDF)
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Dearolf, Charles R
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University of Southern California
Schools of Arts and Sciences
Los Angeles
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Ostrow, A Zachary; Kalhor, Reza; Gan, Yan et al. (2017) Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae. Proc Natl Acad Sci U S A 114:E2411-E2419
Ostrow, A Zachary; Aparicio, Oscar M (2017) Identification of Fkh1 and Fkh2 binding site variants associated with dynamically bound DNA elements including replication origins. Nucleus 8:600-604
Peace, Jared M; Villwock, Sandra K; Zeytounian, John L et al. (2016) Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase. Genome Res 26:365-75
Ostrow, A Zachary; Viggiani, Christopher J; Aparicio, Jennifer G et al. (2015) ChIP-Seq to Analyze the Binding of Replication Proteins to Chromatin. Methods Mol Biol 1300:155-68
Ostrow, A Zachary; Nellimoottil, Tittu; Knott, Simon R V et al. (2014) Fkh1 and Fkh2 bind multiple chromosomal elements in the S. cerevisiae genome with distinct specificities and cell cycle dynamics. PLoS One 9:e87647
Villwock, Sandra K; Aparicio, Oscar M (2014) Two-dimensional agarose gel electrophoresis for analysis of DNA replication. Methods Mol Biol 1205:329-40
Massilamany, Chandirasegaran; Marciano-Cabral, Francine; Rocha-Azevedo, Bruno da et al. (2014) SJL mice infected with Acanthamoeba castellanii develop central nervous system autoimmunity through the generation of cross-reactive T cells for myelin antigens. PLoS One 9:e98506
Peace, Jared M; Ter-Zakarian, Anna; Aparicio, Oscar M (2014) Rif1 regulates initiation timing of late replication origins throughout the S. cerevisiae genome. PLoS One 9:e98501
Aparicio, Oscar M (2013) Location, location, location: it's all in the timing for replication origins. Genes Dev 27:117-28
Pope, Benjamin D; Aparicio, Oscar M; Gilbert, David M (2013) SnapShot: Replication timing. Cell 152:1390-1390.e1

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