RecBCD Fails to Bypass the 5′-To-3′ Single-Stranded DNA Gap After Translocating Along Individual Chi-Containing Duplex DNA

Abstract

The E. coli RecBCD plays an important role of initiating the repair of double-stranded DNA breaks (DSB). Translocating and recognizing chi sequence (5'-GCTGGTGG-3') have been implicated with a conformational change that enables the enzyme to preserve the 3'-to-5' single-stranded DNA for RecA assembly. RecBCD is composed of three subunits, RecB (3'-to-5' helicase), RecD (5'-to-3' helicase), and RecC. Here we used a single-molecule tethered particle motion technique to directly monitor the translocation of RecBCD along chi-contained DNA molecules. Using bead-labeled enzymes, we monitored the RecBCD translocation along individual DNA by measuring the gradual decrease in the bead Brownian motion as the enzyme moves along the DNA towards the surface. DNA substrates were designed that RecBCD would encounter a single-stranded DNA gap after the recognition of the chi sequence. While translocating along chi-free DNA substrates, the time traces showed no apparent pause, which neither 3'-to-5' nor 5'-to-3' ssDNA gap influents the movement of the enzyme1. However, over 50% of RecBCD enzymes failed to pass through the 5'-to-3' ssDNA gap after translocating over chi-containing duplex DNA. Considering RecD as a major 5'-to-3' ssDNA translocase in the RecBCD complex, our observation is consistent with the model that the conformation change occurs after chi recognition and RecD is disengaged from the 5'-ssDNA.(1) Chung, C.; Li, H. W. J. Am. Chem. Soc. 2013, 135, 8920.