Maintenance of genome integrity is the major biological role of RecQ-family helicases via their participation in homologous recombination (HR)-mediated DNA repair processes. RecQ helicases exert their functions by using the free energy of ATP hydrolysis for mechanical movement along DNA tracks (translocation). In addition to the importance of translocation per se in recombination processes, knowledge of its mechanism is necessary for the understanding of more complex translocation-based activities, including nucleoprotein displacement, strand separation (unwinding), and branch migration. Here, we report the key properties of the ssDNA translocation mechanism of Escherichia coli RecQ helicase, the prototype of the RecQ family. We monitored the presteady-state kinetics of ATP hydrolysis by RecQ and the dissociation of the enzyme from ssDNA during single-round translocation. We also gained information on the translocation mechanism from the ssDNA length dependence of the steady-state ssDNA-activated ATPase activity. We show that RecQ occludes 18 ± 2 nt on ssDNA during translocation. The hydrolysis of ATP is noncooperative in the presence of ssDNA, indicating that RecQ active sites work independently during translocation. In the applied conditions, the enzyme hydrolyzes 35 ± 4 ATP molecules per second during ssDNA translocation. RecQ translocates at a moderate processivity, with a mean run length of 100-320 nt on ssDNA. The determined tight mechanochemical coupling of 1.1 ± 0.2 ATP consumed per nucleotide traveled indicates an inchworm-type mechanism.H elicases are NTP-driven nucleic acid motors that couple the liberation of chemical energy to mechanical movement along nucleic acid strands to exert diverse biological activities. Escherichia coli RecQ helicase is the prototype of its family [belonging to superfamily (SF) 2], whose members are essential in genome maintenance from bacteria to humans (1-3) by playing roles in homologous recombination (HR)-mediated dsDNA break (DSB) repair (4-7). In humans, loss-of-function mutations of at least three RecQ helicase homologs are linked to diseases (mostly Bloom, Werner, and Rothmund-Thomson syndromes), which are generally characterized by genome instability resulting in cancer predisposition and premature aging (8). E. coli RecQ suppresses illegitimate recombination (9) and regulates HR-mediated DSB repair via initiating HR and processing HR intermediates (2). Moreover, RecQ stabilizes stalled replication forks as part of the RecF pathway (10, 11).Different types of translocation mechanisms were proposed for SF1 and SF2 helicases. An inchworm mechanism was proposed for bacterial PcrA (12, 13), UvrD (14), and human RecQfamily Bloom syndrome (BLM) (15) helicases, which perform unidirectional translocation based on the consumption of one ATP per kinetic cycle (dictated by a single rate-limiting step), leading to a single nucleotide translocation along ssDNA. A more complex, nonuniform inchworm stepping mechanism was suggested for UvrD (16), NS3 (17), and RecBCD (18) helicase...