Unrepaired DNA breaks can lead to genomic instability or cell death. For repair by the ubiquitous homologous recombination pathway, broken ends are first processed to produce a 3'-ssDNA overhang. In Bacillus subtilis, this reaction is catalysed by AddAB helicase-nuclease complexes; motor proteins that unwind the DNA duplex and degrade the nascent single-strands in a manner regulated by specific single-stranded DNA sequences called Chi recombination hotspots (Yeeles and Dillingham, 2007; Yeeles et al., 2011). We have used Magnetic Tweezers to investigate the real-time dynamics of AddAB translocation on dsDNA and the effect of recombination hotspot recognition on this process. AddAB translocation traces showed a complex appearance with variable velocities between 200-400 bp/s at room temperature. We found that AddAB was prone to stochastic pausing in areas which contained many Chi-like sequences. Experiments using an AddAB mutant that is unable to recognize Chi strongly suggest that this pausing is due to transient recognition of Chi-like sequences and highlight the antagonistic nature of DNA translocation and sequence specific DNA recognition activities. Experiments using substrates containing bona fide Chi sequences showed that AddAB also pauses at Chi, but these events are longer and not exponentially distributed, suggesting a multistep process. We propose a model for the recognition of Chi and Chi-like sequences to explain the origins of this pausing behavior during failed or successful hotspot recognition.Yeeles, J. T., and Dillingham, M. S. (2007). A dual-nuclease mechanism for DNA break processing by AddAB-type helicase-nucleases. J Mol Biol 371, 66-78.Yeeles, J. T., van Aelst, K., Dillingham, M. S., and Moreno-Herrero, F. (2011). Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB helicase-nuclease. Molecular Cell 42, 806-816.