Experiments in this grant focus on sites used for initiation of chromosomal DNA replication using the lower eukaryote S. cerevisiae as a model system. ARSs (autonomously replicating sequences) are putative initiation sites for DNA replication recognized by their ability to support the self-replication of plasmid DNAs. The behavior of 14 different ARSs will be examined during mitosis and meiosis. For the mitotic studies, the presence and copy number of ARS plasmids will be monitored by a visual assay sensitive to plasmid copy number. These studies will provide a detailed description of the efficiency with which an individual ARS promotes the inheritance of plasmid DNAs during mitosis and meiosis. It will also be determined if trans-acting factors encoded by 2 Mum DNA can effect the mitotic stability of either synthetic or authentic yeast chromosomes. DNA contained within small replication bubbles and therefore, by definition, containing an origin of DNA replication, will be isolated from yeast chromosomal DNA after selective labelling with 4-thiouridine. Single-stranded DNA containing thio-substituted residues will be purified by affinity chromatography and used to screen a library of yeast DNA. If this origin-enriched DNA is enriched in ARSs, the experiment will establish a correlation between ARSs and replication origins in yeast chromosomes. Regardless of the outcome in terms of ARSs, the procedure will be valuable for isolating early replicating sequences which can be used in nuclear matrix experiments. The influence of nuclear sub-structure on the organization of yeast DNA, especially in terms of DNA replication, will be examined. Low salt extraction procedures will be developed to test if association of replication forks with the nuclear matrix is observed with different isolation methods. The interaction of replication origins with the nuclear matrix will be examined in populations of cells arrested at different points in the cell cycle: these experiments will determine if origins are permanently or transiently associated with the matrix. As a longer range goal, the association of other structural DNAs with the matrix will be examined. The experiments in this proposal will provide insights into mechanisms involved in control of entry into S-phase and the establishment of a temporal program for chromosome replication. Detailed knowledge of these events is important for understanding how they can be perturbed in abnormal cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM026938-07A1
Application #
3274399
Study Section
Genetics Study Section (GEN)
Project Start
1979-07-01
Project End
1990-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
7
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
075524595
City
Seattle
State
WA
Country
United States
Zip Code
98109
Tran, Phong Lan Thao; Pohl, Thomas J; Chen, Chi-Fu et al. (2017) PIF1 family DNA helicases suppress R-loop mediated genome instability at tRNA genes. Nat Commun 8:15025
Geronimo, Carly L; Zakian, Virginia A (2016) Getting it done at the ends: Pif1 family DNA helicases and telomeres. DNA Repair (Amst) 44:151-158
Phillips, Jane A; Chan, Angela; Paeschke, Katrin et al. (2015) The pif1 helicase, a negative regulator of telomerase, acts preferentially at long telomeres. PLoS Genet 11:e1005186
Lin, Kah Wai; Zakian, Virginia A (2015) 21st Century Genetics: Mass Spectrometry of Yeast Telomerase. Cold Spring Harb Symp Quant Biol 80:111-6
Stundon, Jennifer L; Zakian, Virginia A (2015) Identification of Saccharomyces cerevisiae Genes Whose Deletion Causes Synthetic Effects in Cells with Reduced Levels of the Nuclear Pif1 DNA Helicase. G3 (Bethesda) 5:2913-8
Willis, Nicholas A; Chandramouly, Gurushankar; Huang, Bin et al. (2014) BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks. Nature 510:556-9
Zhou, Ruobo; Zhang, Jichuan; Bochman, Matthew L et al. (2014) Periodic DNA patrolling underlies diverse functions of Pif1 on R-loops and G-rich DNA. Elife 3:e02190
McDonald, Karin R; Sabouri, Nasim; Webb, Christopher J et al. (2014) The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening. DNA Repair (Amst) 24:80-86
Bochman, Matthew L; Paeschke, Katrin; Chan, Angela et al. (2014) Hrq1, a homolog of the human RecQ4 helicase, acts catalytically and structurally to promote genome integrity. Cell Rep 6:346-56
Sabouri, Nasim; Capra, John A; Zakian, Virginia A (2014) The essential Schizosaccharomyces pombe Pfh1 DNA helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage. BMC Biol 12:101

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