Shuttling of RecQ helicase HIM-6 coordinates iterative cycle of unwinding, pulling, and backsliding.

RecQ DNA helicases are a highly conserved family of proteins essential for maintaining genome stability. Despite smFRET studies on repetitive DNA unwinding by RecQ using fluorophore-labeled DNA substrates, the domains controlling this behavior, direct visualization of RecQ movement, and in vivo factors such as protein partners and nucleotide modifications affecting it have remained elusive. Using fluorescently labeled HIM-6 fragments and various DNA substrates, we present the sequential functional activities of HIM-6 and its shuttling along DNA. The helicase domain and zinc-binding element together constitute a minimal functional unit responsible for repetitive unwinding, whereas larger fragments containing additional domains exhibited enhanced DNA unwinding activity and additionally acquired a new strand-pulling activity. During strand pulling, HIM-6 remains stationary and subsequently undergoes backsliding. These three activities occur in an iterative manner and coordinates shuttling of HIM-6 along the DNA. Notably, upon encountering a single ribonucleotide in DNA, HIM-6 paused unwinding, transitioned to a pulling mode, and subsequently pulled in the displaced strand, representing a novel and previously unrecognized trigger for an activity switching. Together, these findings provide new insights into the dynamic behavior of RecQ helicase in regulating genome maintenance.