The accurate and efficient duplication of the eukaryotic genome requires the activation of multiple DNA replication origins distributed over the length of each chromosome. While each origin has the same basic purpose?to direct unwinding of the chromosomal DNA for the initiation of DNA synthesis?each must function within a distinct chromatin context. Despite the well known heterogeneity of chromatin, the field has little understanding of how the core origin binding proteins function within different chromatin contexts or whether origin DNA sequence requirements are influenced by chromatin. The work in the lab of Catherine Fox indicates that these issues are important for explaining why individual origins exhibit differences in how often (origin efficiency) and when (origin activation time) they function during S-phase. This origin functional diversity establishes a temporal pattern to chromosomal replication that is crucial to both cell differentiation and genome stability. The proposed research will define mechanisms that underlie origin functional diversity in the model organism Saccharomyces cerevisiae because the origin-binding proteins and many aspects of chromatin are conserved. In contrast to early models for how origin-specific functional differences in yeast are achieved, the Fox lab proposes that chromatin-mediated regulation of ORC-DNA interactions help establish origin functional diversity. The lab's research provides evidence that ORC-DNA binding in vivo is a target of both positive and negative regulation by chromatin, depending on context; the proposed research will examine the molecular mechanism of two evolutionarily conserved positive regulators. The experiments in this proposal will address a fundamental hypothesis that functionally relevant ORC-origin binding results from a combination of ORC-DNA and ORC-chromatin interactions that result in origin-specific ORC-DNA dynamics that contribute to the regulation of origin function in S-phase.

Public Health Relevance

A defining requirement for life is the timely and accurate reproduction of the cellular genome?even minor perturbations of single steps in this complex, multi-step process can destabilize a cell's genome leading to birth defects, inherited diseases or cancer. Genome duplication requires hundreds of proteins that must work together precisely with each other and with the genome itself. This proposal aims to understand how the conserved proteins that control the first step of genome duplication in eukaryotic cells function and are regulated to promote and maintain healthy cellular life and identity.

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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Carter, Anthony D
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University of Wisconsin Madison
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Hoggard, Timothy A; Chang, FuJung; Perry, Kelsey Rae et al. (2018) Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins. PLoS Genet 14:e1007418
Kuznetsov, Vyacheslav I; Haws, Spencer A; Fox, Catherine A et al. (2018) General method for rapid purification of native chromatin fragments. J Biol Chem 293:12271-12282
Sheets, Michael D; Fox, Catherine A; Dowdle, Megan E et al. (2017) Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development. Adv Exp Med Biol 953:49-82
Dummer, Antoinette M; Su, Zhangli; Cherney, Rachel et al. (2016) Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast. PLoS Genet 12:e1006094
Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra et al. (2016) High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation. G3 (Bethesda) 6:993-1012
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
Hoggard, Timothy; Shor, Erika; Müller, Carolin A et al. (2013) A Link between ORC-origin binding mechanisms and origin activation time revealed in budding yeast. PLoS Genet 9:e1003798
Shor, Erika; Fox, Catherine A; Broach, James R (2013) The yeast environmental stress response regulates mutagenesis induced by proteotoxic stress. PLoS Genet 9:e1003680
Fox, Catherine A; Gartenberg, Marc R (2012) Palmitoylation in the nucleus: a little fat around the edges. Nucleus 3:251-5
Park, Sookhee; Patterson, Erin E; Cobb, Jenel et al. (2011) Palmitoylation controls the dynamics of budding-yeast heterochromatin via the telomere-binding protein Rif1. Proc Natl Acad Sci U S A 108:14572-7

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