In this proposal we focus on the Pif1 sub-family of DNA helicases and test the hypothesis that one of their functions is to facilitate DNA replication across naturally occurring barriers. We showed that DNA-bound Rap1 is a barrier to the strand-displacement DNA synthesis activity of DNA polymerase ? and that Pif1 displaces Rap1, allowing DNA replication across the barrier. We hypothesize that proteins bound to the DNA, at telomeres and non-telomeric sites, are displaced by Pif1 to facilitate DNA replication, and that this activity is fundamental and conserved among Pif1 homologues. G-quadruplexes and DNA hairpins can also be obstacles to DNA replication. Pif1 is thought to facilitate replication-fork progression by unwinding G- quadruplexes. However, little is known about this activity of Pif1 in the presence of single- stranded DNA binding proteins (SSBs). We hypothesize that the activity of Pif1 is needed to facilitate DNA replication across G-quadruplexes when SSBs cannot effectively melt the DNA secondary structure. Furthermore, we hypothesize that Pif1 stimulates DNA synthesis by DNA polymerase ? across DNA hairpins. We showed that unwinding of dsDNA by a Pif1 monomer is balanced by a Pif1-dependent DNA rewinding activity. We hypothesize that under physiological conditions rewinding is inhibited by concomitant synthesis of DNA by polymerases. Mutations of Pif1 that affect the balance between unwinding and rewinding will allow us to determine the origin of this newly discovered property. To test these hypotheses, we propose three Aims: 1) determine whether protein obstacles that impede DNA replication are displaced by Pif1; 2) determine whether DNA secondary structures are unwound by Pif1 to facilitate DNA replication; 3) determine the mechanism underlying the Pif1-dependent DNA rewinding activity.

Public Health Relevance

The role of DNA helicases in promoting progression of DNA replication through sites that are difficult to replicate is of particular importance for our understanding of maintenance of genomic stability. During each cell cycle, DNA replication needs to overcome multiple obstacles, such as proteins tightly bound to DNA, DNA secondary structures, and damaged DNA. The long-term goal of our research is to understand the activity of accessory helicases at such obstacles in general, but particularly at telomeres, which are known to impede replication and whose integrity is fundamental to genome stability.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function A Study Section (MSFA)
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Reddy, Michael K
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Washington University
Schools of Medicine
Saint Louis
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Singh, Saurabh P; Kukshal, Vandna; De Bona, Paolo et al. (2018) The mitochondrial single-stranded DNA binding protein from S. cerevisiae, Rim1, does not form stable homo-tetramers and binds DNA as a dimer of dimers. Nucleic Acids Res 46:7193-7205
Dahan, Danielle; Tsirkas, Ioannis; Dovrat, Daniel et al. (2018) Pif1 is essential for efficient replisome progression through lagging strand G-quadruplex DNA secondary structures. Nucleic Acids Res 46:11847-11857
Geronimo, Carly L; Singh, Saurabh P; Galletto, Roberto et al. (2018) The signature motif of the Saccharomyces cerevisiae Pif1 DNA helicase is essential in vivo for mitochondrial and nuclear functions and in vitro for ATPase activity. Nucleic Acids Res 46:8357-8370
Koc, Katrina N; Singh, Saurabh P; Stodola, Joseph L et al. (2016) Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase ?. Nucleic Acids Res 44:3811-9
Singh, Saurabh P; Koc, Katrina N; Stodola, Joseph L et al. (2016) A Monomer of Pif1 Unwinds Double-Stranded DNA and It Is Regulated by the Nature of the Non-Translocating Strand at the 3'-End. J Mol Biol 428:1053-1067
Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto et al. (2016) Chemo-mechanical pushing of proteins along single-stranded DNA. Proc Natl Acad Sci U S A 113:6194-9
Feldmann, Erik A; De Bona, Paolo; Galletto, Roberto (2015) The wrapping loop and Rap1 C-terminal (RCT) domain of yeast Rap1 modulate access to different DNA binding modes. J Biol Chem 290:11455-66
Koc, Katrina N; Stodola, Joseph L; Burgers, Peter M et al. (2015) Regulation of yeast DNA polymerase ?-mediated strand displacement synthesis by 5'-flaps. Nucleic Acids Res 43:4179-90
Feldmann, Erik A; Koc, Katrina N; Galletto, Roberto (2015) Alternative arrangements of telomeric recognition sites regulate the binding mode of the DNA-binding domain of yeast Rap1. Biophys Chem 198:1-8
Feldmann, Erik A; Galletto, Roberto (2014) The DNA-binding domain of yeast Rap1 interacts with double-stranded DNA in multiple binding modes. Biochemistry 53:7471-83

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