The long term goal of this grant is to contribute to the understanding of mechanisms responsible for the faithful replication of eukaryotic chromosomes, using yeasts as models. The four aims concern three members of the Pif1 family of DNA helicases, a virtually ubiquitous eukaryotic helicase family that is named for its prototypical member the S. cerevisiae Pif1.
The first aim will determine how the S. cerevisiae Pif1 inhibits telomerase- mediated telomere extension by asking if Pif1 inhibits the frequency and/or extent of telomere lengthening and to determine if its activity is regulated by telomere length.
The second aim extends our recent discovery that the S. cerevisiae Pif1 is a potent unwinder of G-quadruplex DNA (G4), very stable four-stranded DNA structures that are held together by G-G base pairs. We will use genome wide approaches to identify Pif1 binding sites and sites of DNA damage in Pif1-depeleted cells to see if Pif1 acts preferentially at G4 sites. A variety of genetic and physical assays will be used to determine if sequences that can form G4 structures in vitro are selectively destabilized in pif1 mutant cells and if so, if this instability is correlated with fork stalling and/or breakage at G4 sites. These experiments will help resolve controversies as to whether G4 structures form in vivo.
The third aim will continue our studies to determine the essential role(s) of the S. pombe Pfh1 helicase in chromosome replication. To identify Pfh1 targets, we will again use genome wide approaches, which will identify Pfh1 binding sites, as well as sites of chromosome breakage upon Pfh1 depletion. Physical and genetic assays will be used to determine if Pfh1 depletion results in fork stalling and/or breakage at hard to replicate sites and/or at sites identified in the genome wide analyses. Pfh1 interacting proteins will be identified by mass spectrometry with the goal that their identities will help determine the specific DNA transactions in which Pfh1 is engaged.
The fourth aim describes work to continue our analysis of the S. cerevisiae Rrm3 helicase, which has the unique property of promoting fork progression through stable protein complexes. To circumvent difficulties purifying full length Rrm3 for in vitro studies, we will use a genetic strategy to identify mutations in the amino terminus of Rrm3 that render it easy to purify without affecting its in vivo functions. We will use purified Rrm3 to determine its preferred nucleic acid substrates and to test its ability to displace protein complexes from DNA in vitro. A genetic approach is described to identify proteins that either assist Rrm3 or substitute for it during replication through stable protein complexes. Because helicases are essential for DNA replication, repair and recombination, it is not surprising that their mutation can lead to human diseases characterized by genome instability, such as premature aging, and cancer. In yeasts, Pif1 family proteins have important and so far unique properties in DNA replication. These studies will lay the groundwork for the identification and analysis of human proteins with similar functions.

Public Health Relevance

DNA helicases have critical roles in DNA replication, repair and recombination. Therefore, it is not surprising that their mutation can lead to genome instability and inherited human diseases such as cancer and premature aging. This grant describes experiments to determine the mechanisms by which the eukaryotic Pif1 family helicases, which have unique properties in chromosome replication, contribute to genome stability. These studies will lay the groundwork for the identification and analysis of human proteins with similar functions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026938-33
Application #
8197546
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Carter, Anthony D
Project Start
1979-07-01
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
33
Fiscal Year
2012
Total Cost
$608,286
Indirect Cost
$227,379
Name
Princeton University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08544
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
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
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) 24:80-6
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
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
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
Paeschke, Katrin; Bochman, Matthew L; Garcia, P Daniela et al. (2013) Pif1 family helicases suppress genome instability at G-quadruplex motifs. Nature 497:458-62
Wu, Yun; DiMaggio Jr, Peter A; Perlman, David H et al. (2013) Novel phosphorylation sites in the S. cerevisiae Cdc13 protein reveal new targets for telomere length regulation. J Proteome Res 12:316-27
Webb, Christopher J; Wu, Yun; Zakian, Virginia A (2013) DNA repair at telomeres: keeping the ends intact. Cold Spring Harb Perspect Biol 5:

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