Direct Observation of Recbcd Helicase as ssDNA Translocases

Abstract

The E. coli RecBCD is a heterotrimeric enzyme composed of two helicase motors with different polarities: RecB (3’-to-5’) and RecD (5’-to-3’). This Superfamily I helicase is responsible for the initiation of 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) without secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt duplex DNA, and translocate along the duplex/single-stranded/duplex DNA substrate, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over long ssDNA gap in either 3’-to-5’ or 5’-to-3’ direction indicates that both RecB and RecD are ssDNA translocases. We also applied continuous force (∼ 0.2 pN) to stretch DNA substrates and to remove any potentially transient looped ssDNA structure, and observed continuous translocation of RecBCD. It confirms the ssDNA translocase activities of RecBCD helicase. About 78 % of active tethers showed full translocation across the ssDNA to the dsDNA region, and the other 22 % enzymes dissociated from the ss/dsDNA junction after translocating across the ssDNA region. We also prepared a double-gapped substrate containing two regions of ssDNA with opposite polarities (5’-to-3’ and 3’-to-5’) intermitted by duplex DNA. RecBCD was able to translocate across both ssDNA regions in either ssDNA orientation orders, with 20 - 40 % of tethers dissociating while entering the second ssDNA region. These results suggest a mechanism that RecBCD is able to switch motors and rethread into the other strand after translocating along an ssDNA gapped region.