Translocation step size and mechanism of theRecBC DNA helicase

DNA helicases are ubiquitous enzymes that unwind double-stranded DNA(1-3). 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 nucle oside triphosphate hydrolysis to both translocation and double-stranded DNA unwinding to produce separate strands of DNA. The RecBC enzyme is a proces sive DNA helicase that functions in homologous recombination in Escherichia coli; it unwinds up to 6,250 base pairs per binding event and hydrolyses s lightly more than one ATP molecule per base pair unwound. Here we show, by using a series of gapped oligonucleotide substrates, that this enzyme trans locates 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 i s much larger than the amount of double-stranded DNA that can be unwound us ing the free energy derived from hydrolysis of one molecule of ATP, implyin g that translocation and DNA unwinding are separate events. We propose that the RecBC enzyme both translocates and unwinds by a quantized, two-step, i nchworm-like mechanism that may have parallels for translocation by other l inear motor proteins.