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    Awarded Grants
    Maintenance of Genome Stability and Telomere Length by Pfh1p DNA Helicase in Schizosaccharomyces Pombe

    Scientific Abstract:
    Maintenance of genome stability and telomere length by Pfh1p DNA helicase in Schizosaccharomyces pombe a.) Background: Genomic instability is prevalent both as a cause and as a consequence of cancer. DNA helicases play critical roles in DNA replication, recombination and repair and their importance in maintaining genome stability is underscored by the fact that mutations in the BLM, WRN and RECQ4 helicases result in Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome, respectively, all of which are human genetic disorders with genomic instability and a predisposition to cancer. The Pif1p subfamily of DNA helicases is well conserved from yeast to human and our lab has demonstrated that the two Pif1p subfamily members in Saccharomyces cerevisiae, Pif1p and Rrm3p, exert important functions in the maintenance of genome stability and telomere length regulation: Rrm3p prevents replication fork collapse and subsequent chromosome breakage by facilitating replication fork progression and resolution of converged replication forks. Pif1p acts as a catalytic inhibitor of telomerase. The only other member of the Pif1p subfamily studied to date is Pfh1p, an essential 5’ 3’ DNA helicase in Schizosaccharomyces pombe, which like humans contains only one Pif1p-like helicase. The telomeres, centromeres and replication origins of this genetically tractable model organism closely resemble their human counterparts. Thus, S. pombe offers a unique opportunity to study the functions of the Pif1p subfamily of helicases. Preliminary evidence suggests that Pfh1p functions in maintenance of both genome stability and telomere length. Cells depleted for functional Pfh1p arrest in late S phase in a checkpoint-dependent manner, and two cold-sensitive alleles of Pfh1p confer extreme sensitivity to DNA damaging drugs. Furthermore, overexpression of Pfh1p results in telomere lengthening, while depletion of Pfh1p results in telomere shortening. b.) Objective/Hypothesis: I will test the hypothesis that Pfh1p acts to maintain genome stability and telomere length by exerting the following functions: 1. Facilitating replication fork progression and resolution of converged forks, 2. Promoting recombinational repair, 3. Promoting telomerase activity. To this end, I will characterize these functions as outlined in my specific aims: c., d.) Specific Aims and Study Design: 1. To determine whether Pfh1p acts in facilitating replication fork progression and resolving converged replication forks at the telomeres, rDNA and mating type loci, I will analyze replication intermediates by two dimensional gel electrophoresis. 2. To determine whether Pfh1p functions in recombinational repair, I will analyze the contribution of Pfh1p to recombinational repair using marker loss assays in a strain with a single and inducible double-strand break 3. To determine whether Pfh1p increases association of Trt1p, the catalytic subunit of telomerase, with the telomere in vivo, I will perform chromatin immuno-precipitations (ChIP) 4. To determine whether Pfh1p increases the processivity of telomerase in vitro I will perform primer extension assays with fractionated telomerase holoenzyme These experiments will be carried out in a strain with an inducible promoter driving Pfh1p expression. This will allow comparison between cells containing low, wild-type and high levels of Pfh1p. In addition, the same experiments will be conducted in strains with cold-sensitive alleles of Pfh1p shifted from the permissive to the restrictive temperature. e.) Potential Outcomes and Benefits of the Research: Understanding how helicases increase the fidelity and efficiency of DNA replication, recombination and repair to maintain genome stability is crucial to understanding cancer initiation. Furthermore, as 90% of all cancer types, including 75% of breast cancers, contain telomerase activity, telomerase may be a potential target for anti-cancer therapies. Identifying proteins that regulate telomerase activity, may broaden the list of such potential targets significantly.

    Lay Abstract:
    Maintenance of genome stability and telomere length by Pfh1p DNA helicase in Schizosaccharomyces pombe a.) Background: Our cells store hereditary information in the genome, an assembly of many genes contained in linear DNA molecules called chromosomes. The term genomic instability refers to events that significantly change the content of our genome, for example by deletion of genes. When these changes lead to loss of genes that restrict cell growth and division, the cell has taken a crucial step to giving rise to a tumor. DNA replication, the duplication of the genome before every cell division, depends on many enzymes to ensure speed and accuracy. However when the genes encoding for these enzymes are defective or mutated, DNA replication can lead to genomic instability. One important class of such enzymes are DNA helicases, which separate the two complementary DNA strands of each chromosome to allow DNA replication. Mutations in many helicases are known to lead to several disorders that cause genomic instability and predisposition to cancer. Telomeres are the physical ends of chromosomes. Telomeres shorten with every cell division and the shorter they get the less likely a cell is able to continue cell growth and division. Cancer cells have to go through many divisions to form a tumor and in order to keep their telomeres long they activate an enzyme called telomerase that lengthens telomeres when they get too short. The Pif1p-like helicases are found in yeasts and all animals up to humans. Our lab has demonstrated that two of these helicases in baker’s yeast, Pif1p and Rrm3p, exert important functions in the maintenance of genome stability and telomere length regulation: Rrm3p facilitates the progression of replication enzymes that travel along each DNA strand as they copy all genes onto a second DNA strand. If they have to stop, the DNA could break, which is an initiating event for genome instability if not repaired correctly. Pif1p inhibits telomerase to keep telomeres at a normal length. The only other Pif1p-like helicase that has been studied to date is Pfh1p in the fission yeast, which has chromosomes very similar to human chromosomes. Preliminary evidence suggests that Pfh1p functions in maintenance of both genome stability and telomere length. Cells without Pfh1p stop dividing and cannot repair their DNA when treated with DNA damaging drugs. High levels of Pfh1p result in longer telomeres, and low levels of Pfh1p result in shorter telomeres. b.) Objective/Hypothesis: I will test the hypothesis that Pfh1p acts to maintain genome stability and telomere length by exerting the following functions: 1. Facilitating the progression of replication enzymes 2. Promoting DNA repair, 3. Promoting telomerase activity. To this end, I will characterize these functions as outlined in my specific aims: c., d.) Specific Aims and Study Design: 1. To determine whether Pfh1p acts in facilitating the progression of other replication enzymes, I will analyze the abundance and structure of specific DNA molecules during replication 2. To determine whether Pfh1p functions in DNA repair, I will analyze the contribution of Pfh1p to the repair of a single chromosome break 3. To determine whether Pfh1p increases the likelihood of telomerase to lengthen the telomeres, I will determine how well telomerase is associating with the telomeres 4. To determine whether Pfh1p increases the efficiency of telomerase I will determine how well telomerase lengthens artificial telomeres. These experiments will be carried out in a strain with low, normal and high levels of Pfh1p, to determine how Pfh1p contributes to the results in each experiment. e.) Potential Outcomes and Benefits of the Research: Understanding how helicases increase the fidelity and efficiency of DNA replication, recombination and repair to maintain genome stability is crucial to understanding cancer initiation. Furthermore, as 90% of all cancer types, including 75% of breast cancers, contain telomerase activity, telomerase may be a potential target for anti-cancer therapies. Identifying proteins that regulate telomerase activity, may broaden the list of such potential targets significantly.