Eukaryotic chromosomes are replicated and segregated with extremely high fidelity. The long range goal of this grant is to identify the mechanisms that ensure the accuracy of these processes, using S. cerevisiae as a model system.
The specific aims for this funding period concern telomeric and sub-telomeric regions of the chromosome. Telomeres were long thought to be essential for the stable maintenance and complete replication of eukaryotic chromosome. However, studies in Drosophila demonstrate that chromosome without telomeres are not degraded nor do they fuse with other broken ends (8,68). In contrast, indirect experiments in other systems suggest that chromosomes without telomeres are unstable. To determine the precise role of telomeres in yeast, we will eliminate the telomeric repeats from an authentic, but totally dispensable, chromosome in a strain where broken chromosomes cannot be repaired by conventional recombination. In addition to the simple repeats found at the very ends of chromosomes, sub-telomeric regions of most eukaryotic chromosomes have middle repetitive DNAs called telomere-associated (TA) sequences. To determine the function of TA sequences, derivatives of chromosome VII that lack TA sequences on both arms have been constructed. We will determine the stability of these deleted chromosomes in mitosis, meiosis, and after X-ray induced breakage. In virtually all eukaryotes, telomeres are dynamic structures. Almost surely recombination in telomeric and sub-telomeric regions contributes to variability. work from our lab provided the first evidence for telomere- telomere recombination by demonstrating that foreign telomeric sequences recombine in yeast by a novel mechanism that requires very little homology (93,128). To determine the function of this recombination, we will use a system where telomere-telomere recombination can be detected between """"""""natural"""""""" yeast telomeres on plasmids and chromosomes and have designed schemes to identify mutants in telomere-telomere recombination. A possible role for telomere recombination is to assist in the replication of chromosome ends. Using two dimensional gel electrophoresis of plasmid DNA from synchronized cells, we have identified a presumptive intermediate in telomere replication, the first in vivo intermediate identified in any system. We describe experiments to continue analysis of its structure, to determine the gene products on which its synthesis depends, to determine if replication of chromosomal telomeres proceeds by the same mechanism, and to understand how its interactions in vivo contribute to processes like telomere recombination. Aneuploidy and chromosome rearrangements are associated with virtually human cancers, with aging, and with birth defects. Changes in telomeric and sub-telomeric regions can generate the kinds of chromosome abnormalities that are associated with these diseases. Given that the telomeric regions of chromosomes in yeast and humans are similarly organized, a molecular and genetic understanding of the mechanisms that underly these events in yeast will contribute to the understanding of the genesis of chromosome abnormalities in higher cells.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Fred Hutchinson Cancer Research Center
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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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