<span class="paragraphSection"><div class="boxTitle">Abstract</div>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 <span style="font-style: italic;">in vivo</span> 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.</span>