DNA helicases are ubiquitous enzymes that unwind double-stranded DNA. They are a diverse group of proteins that move in a linear fashion along a one-dimensional polymer lattice - DNA - by using a mechanism that couples nucleoside triphosphate hydrolysis to both translocation and double stranded DNA unwinding to produce separate strands of DNA. The RecBC enzyme is a processive DNA helicase that functions in homologous recombination in Escherichia coli; it unwinds up to 6,250 base pairs per binding event and hydrolyses slightly more than one ATP molecule per base pair unwound. Here we show, by using a series of gapped oligonucleotide substrates, that this enzyme translocates along only one strand of duplex DNA in the 3' → 5' direction. The translocating enzyme will traverse, or 'step' across, single- stranded DNA gaps in defined steps that are 23 (±2) nucleotides in length. This step is much larger than the amount of double-stranded DNA that can be unwound using the free energy derived from hydrolysis of one molecule of ATP, implying that translocation and DNA unwinding are separate events. We propose that the RecBC enzyme both translocates and unwinds by a quantized, two-step, inchworm-like mechanism that may have parallels for translocation by other linear motor proteins.
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