Pif1 family helicases are conserved from bacteria to humans. Here, we report a 
novel 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: 
http://dx.doi.org/10.7554/eLife.02190.001.