title: Direct observation of RecBCD helicase as single-stranded DNA translocases.
abstract: The heterotrimeric Escherichia coli RecBCD enzyme comprises two helicase motors with different polarities: RecB (3'-to-5') and RecD (5'-to-3'). This superfamily I helicase is responsible for initiating DNA double-strand-break (DSB) repair in the homologous recombination pathway. We used single-molecule tethered particle motion (TPM) experiments to visualize the RecBCD helicase translocation over long single-stranded (ss) DNA (>200 nt) with no apparent secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt-end DNA, and translocate along the DNA substrates containing an ssDNA gap, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over a long gap in either 3'-to-5' or 5'-to-3' ssDNA direction indicates that RecBCD helicase possesses ssDNA translocase activities in both polarities. Most RecBCD active tethers showed full translocation across the ssDNA to the dsDNA region, with about 19% of enzymes dissociated from the ss/dsDNA junction after translocating across the ssDNA region. In addition, we prepared DNA substrates containing two opposite polarities (5'-to-3' and 3'-to-5') of ssDNA regions intermitted by duplex DNA. RecBCD was able to translocate across both ssDNA regions in either ssDNA orientation orders, with less than 40% of tethers dissociating when entering into the second ssDNA region. These results suggest a model that RecBCD is able to switch between ssDNA translocases and rethread the other strand of ssDNA.
authors.affiliation: Department of Chemistry, National Taiwan University, Taiwan.
authors.forename: Cinya
authors.initials: C
authors.lastname: Chung
authors.forename: Hung-Wen
authors.initials: HW
authors.lastname: Li
mesh_terms: D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D043211:Exodeoxyribonuclease V; D004265:DNA Helicases