Single-stranded DNA (ssDNA) and RNA regions that include at least four closely 
spaced runs of three or more consecutive guanosines strongly tend to fold into 
stable G-quadruplexes (G4s). G4s play key roles as DNA regulatory sites and as 
kinetic traps that can inhibit biological processes, but how G4s are regulated 
in cells remains largely unknown. Here, we developed a kinetic framework for G4 
disruption by DEAH-box helicase 36 (DHX36), the dominant G4 resolvase in human 
cells. Using tetramolecular DNA and RNA G4s with four to six G-quartets, we 
found that DHX36-mediated disruption is highly efficient, with rates that depend 
on G4 length under saturating conditions (kcat) but not under subsaturating 
conditions (kcat/Km ). These results suggest that a step during G4 disruption 
limits the kcat value and that DHX36 binding limits kcat/Km Similar results were 
obtained for unimolecular DNA G4s. DHX36 activity depended on a 3' ssDNA 
extension and was blocked by a polyethylene glycol linker, indicating that DHX36 
loads onto the extension and translocates 3'-5' toward the G4. DHX36 unwound 
dsDNA poorly compared with G4s of comparable intrinsic lifetime. Interestingly, 
we observed that DHX36 has striking 3'-extension sequence preferences that 
differ for G4 disruption and dsDNA unwinding, most likely arising from 
differences in the rate-limiting step for the two activities. Our results 
indicate that DHX36 disrupts G4s with a conventional helicase mechanism that is 
tuned for great efficiency and specificity for G4s. The dependence of DHX36 on 
the 3'-extension sequence suggests that the extent of formation of genomic G4s 
may not track directly with G4 stability.