Atomic Force Microscopy Shows that Chi Sequences and SSB Proteins Prevent DNA Reannealing Behind the Translocating AddAB Helicase-Nuclease

Abstract

Recombinational repair of DNA breaks requires processing of a DNA end to a 3′-ssDNA overhang. In B.subtilis, this task is done by the helicase-nuclease AddAB which generates ssDNA overhangs terminated at a recombination hotspot (Chi) sequence. This is a substrate for the formation of a RecA nucleoprotein filament that searches for a homologous donor molecule and catalyses DNA strand exchange to promote repair. In this study, we have used AFM to visualize the products of reactions including AddAB and double-stranded DNA molecules. AFM images consistently showed a remaining population of apparently unprocessed dsDNA molecules. The fraction of unprocessed molecules dropped upon addition of increasing concentrations of SSB protein or larger amounts of AddAB protein. Moreover, a larger fraction of DNA molecules were processed to ssDNA when DNA substrates contained the regulatory Chi sequence. Our results are consistent with a model in which the DNA strands reanneal behind the translocating AddAB enzyme. This effect is suppressed by destabilizing the interaction between DNA strands via binding of SSB or multiple AddAB motors, or by the interaction between AddAB and Chi during translocation.