Periodic DNA patrolling underlies diverse functions of Pif1 on R-loops and G-rich DNA

RESEARCH ARTICLE elifesciences.org Periodic DNA patrolling underlies diverse functions of Pif1 on R-loops and G-rich DNA Ruobo Zhou1†, Jichuan Zhang1,2, Matthew L Bochman3‡, Virginia A Zakian3, Taekjip Ha1,4* Center for the Physics of Living Cells, Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States; 2Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, United States; 3Department of Molecular Biology, Princeton University, Princeton, United States; 4Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, United States 1 Abstract Pif1 family helicases are conserved from bacteria to humans. Here, we report a novel *For correspondence: tjha@ illinois.edu Present address: †Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States; ‡ Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, United States Competing interests: The authors declare that no competing interests exist. Funding: See page 14 Received: 02 January 2014 Accepted: 17 March 2014 Published: 29 April 2014 Reviewing editor: Johannes Walter, Harvard Medical School, United States Copyright Zhou et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. DNA patrolling activity which may underlie Pif1’s diverse functions: a Pif1 monomer preferentially anchors itself to a 3′-tailed DNA junction and periodically reel in the 3′ tail with a step size of one nucleotide, extruding a loop. This periodic patrolling activity is used to unfold an intramolecular G-quadruplex (G4) structure on every encounter, and is sufficient to unwind RNA-DNA heteroduplex but not duplex DNA. Instead of leaving after G4 unwinding, allowing it to refold, or going beyond to unwind duplex DNA, Pif1 repeatedly unwinds G4 DNA, keeping it unfolded. Pif1-induced unfolding of G4 occurs in three discrete steps, one strand at a time, and is powerful enough to overcome G4-stabilizing drugs. The periodic patrolling activity may keep Pif1 at its site of in vivo action in displacing telomerase, resolving R-loops, and keeping G4 unfolded during replication, recombination and repair. DOI: 10.7554/eLife.02190.001 Introduction Approximately, 1% of eukaryotic genes encode DNA or RNA helicases. These enzymes function in nearly all aspects of nucleic acid metabolism in living organisms (Singleton et al., 2007; Lohman et al., 2008). Although helicases were originally recognized as enzymes that catalyze the strand separation of double-stranded nucleic acids, it is now evident that helicases, as defined by a series of characteristic sequence motifs, may have additional functions aside from unwinding duplexes, including protein displacement from DNA and RNA, the unwinding of G-quadruplex (G4) structures, remodelling of chromatin and ribonucleoprotein complexes, promotion of Holliday junction branch migration and the catalysis of a range of nucleic acid conformational changes (Singleton et al., 2007; Lohman et al., 2008; Pyle, 2008; Paeschke et al., 2013). The Pif1 DNA helicase is an example of a multi-functional helicase. The Pif1 helicase family is a group of 5′→3′ directed, ATP-dependent, super-family (SF) 1B helicases that are evolutionarily conserved from bacteria to humans (Bochman et al., 2010; Paeschke et al., 2013). The Saccharomyces cerevisiae Pif1 helicase (Pif1), the prototypical member of the Pif1 helicase family, plays critical roles in inhibiting telomerase activity at telomeres and double-stranded DNA breaks (DSBs), processing Okazaki fragments, promoting break-induced replication, maintaining mitochondrial DNA, and preventing replication pausing and DSBs at G-quadruplex (G4) motifs (Boule and Zakian, 2006; Bochman et al., 2010; Lopes et al., 2011; Paeschke et al., 2011, 2013; Wilson et al., 2013). However, the molecular mechanisms responsible for these diverse Pif1 functions remain elusive. For Zhou et al. eLife 2014;3:e02190. DOI: 10.7554/eLife.02190 1 of 16 Research article Biophysics and structural biology eLife digest Helicases are enzymes that are best known for their ability to separate the two strands of DNA that make up the famous double-helix structure. Many important processes within cells—including the expression of genes as proteins, and the replication of DNA before cell division—rely on DNA molecules being separated in this way. However, these enzymes can perform many other roles that help maintain the integrity of a cell’s DNA. The genetic code is written using four DNA bases—called A, C, G and T—and if a stretch of DNA contains lots of G bases, then one of the strands can loop back upon itself three times to form a structure known as a ‘G-quadruplex’. These structures can prevent the expression of genes, and slow the replication of DNA. However, a helicase called Pif1 can unwind G-quadruplexes to allow these activities to continue. This helicase is found in many organisms, from bacteria to humans, and carries out multiple functions for a cell. However, the exact mechanisms underlying these activities are unknown. Now, Zhou et al. have used biophysical techniques to reveal that individual Pif1 proteins bind to single-stranded overhangs at one end of a DNA molecule. Pif1 also binds to forks in DNA where the double helix separates into two single strands. And once Pif1 has bound to the DNA, it works to ‘reel in’ the overhang or a single strand, one base at a time. This activity can unwind a G-quadruplex, and individual Pif1 proteins will patrol DNA to keep this structures unwound without unraveling the double helix itself. Separating the two strands of DNA actually needs multiple Pif1 proteins to join and work together. As it patrols, Pif1 also displaces other proteins from DNA and removes unusual, and potentially harmful, structures in DNA (such as RNA molecules that have displaced one of the strands of DNA double helix). The next challenge will be to address important questions that remain unanswered including: how does Pif1 recognize DNA structures and change its activity; and how does it coordinate with other proteins that target the same structures? DOI: 10.7554/eLife.02190.002 example, Pif1 is known to unwind RNA/DNA hybrids better than dsDNA but its mechanistic basis is unknown (Boule and Zakian, 2007). In addition, how Pif1 may selectively function on certain DNA structures such as stalled replication forks or G4 structures is unclear. Here, we show that a Pif1 monomer is preferentially recruited to 3′ ss-dsDNA junctions and induces repetitive DNA looping that is tightly coupled to its translocation activity powered by its ATPase. This periodic DNA patrolling activity of a Pif1 monomer can be used to unwind RNA–DNA hybrids in its path but not DNA–DNA duplexes because DNA unwinding requires the cooperation of multiple (...truncated)