This is one of the many projects started with the group of Dr. Mark Dillingham from University of Bristol in the context of our Starting Grant ERC Project.
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 in 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 project, we have used AFM and Magnetic Tweezers to visualize and characterize the products of reactions including AddAB and double-stranded DNA molecules, and to monitor the real time dynamics of DNA translocation and unwinding.
Our work and that of others have provided an exquisite understanding of the end-processing step for DNA repair by homologous recombination in Bacteria.
University of Bristol: Mark Dillingham Group
Yeeles et al. Molecular Cell 42, 806-816 (2011).
Recombination Hotspots and Single-Stranded DNA Binding Proteins Couple DNA Translocation to DNA Unwinding by the AddAB Helicase-Nuclease
Carrasco et al. PNAS 110 (28), E2562-2571 (2013).
On the mechanism of recombination hotspot scanning during double-stranded DNA break resection
Carrasco et al. DNA Repair 20, 119-129 (2014) (cover article).
Single molecule approaches to monitor the recognition and resection of double-stranded DNA breaks during homologous recombination
Gilhooly et al. Nucleic Acids Research 44(6), 2727-2741 Published Online Jan 13 (2016).
Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination