Final Report Summary - SM-DNA-REPAIR (New single-molecule techniques and their application in the study of DNA break repair) The aims of the SM-DNA-Repair project were two-fold: firstly, to develop novel biophysical instruments for fast Atomic Force Microscopy imaging in liquid and an Optical and Magnetic Tweezers setup; and secondly, to monitor and characterise the real-time dynamics of DNA repair processes using these complementary biophysical approaches. Precisely, we focussed in the AddAB helicase-nuclease and the SMC (Structural Maintenance of Chromosomes) complex from Bacillus subtilis. AddAB is a prototype molecular machine responsible for the long-range resection of double-stranded DNA breaks (DSB) in bacteria. SMC is a protein complex required for chromosome compaction and faithful DNA segregation. We have built a Magnetic Tweezers (MT) machine that can manipulate single DNA molecules and measure force and torque applied by molecular motors. This setup consists of a pair of magnets positioned over a flow cell on an inverted optical microscope. Magnetic beads are used as probes that are manipulated by an external force that pulls them toward the magnets. From optical images acquired with a CCD camera, the position and the force acting on these beads can be calculated. We have used this machine to investigate the dynamics of AddAB translocation and hotspots (Chi) scanning during double-stranded DNA break resection (PNAS, 2013). We have discovered that AddAB is prone to stochastic pausing due to transient recognition of Chi-like sequences unveiling an antagonistic relationship between DNA translocation and sequence-specific recognition. AddAB also paused at bona-fide Chi sequences, but these pauses were non-exponentially distributed suggesting a multi-step mechanism in the process of Chi recognition. We have also built an Optical Tweezers setup to study DNA-protein interactions at the single particle level. It consists of a single trap created with an infrared 5 W laser, the power of which is further stabilized using a feedback routine. The laser trap is able to stably capture micrometer-size beads and to withstand forces up to ~120 pN. The position of the trapped bead is determined with video microscopy and with an additional 15 mW laser by measuring the reflection in the trapped bead with a sensitive photodetector. The system incorporates a nm resolution piezo stage that holds a micropipette where a second bead is attached. With this setup we have been able to meausure force-extension curves on dsDNA and the forces involved in single biotin-strepavidin interactions. A commercial Atomic Force Microscope was modified with the aim of imaging DNA in buffer at about one image per second rate. We have successfully designed and built an AFM head capable of using small cantilevers. This AFM head is able to focus the detection laser beam in a 5-10 µm spot. Additionally, we have incorporated an analogue PID controller to increase the bandwidth of the feedback loop. All these developments allowed us to observe DNA molecules at frame rates of about 1 image per second in air and in buffer. The AFM setup was employed to investigate the products of the AddAB resection reaction. We discovered that AddAB translocation is not coupled to DNA unwinding in the absence of single-stranded DNA binding proteins (Molecular Cell, 2011). However, we found that the recognition of Chi sequences activates unwinding ensuring the downstream formation of single-stranded DNA that is required for RecA-mediated recombinational repair (Molecular Cell, 2011). We have also developed new methodologies to determine the stoichiometry of protein complexes using the AFM technique. These methods are based in precise measurements of the volume of proteins from AFM images using a DNA molecule as a fiducial marker (Biophysical Journal, 2012, Methods, 2013). The stoichiometry of the SMC complex was determined using these novel methods. Finally, our expertise in AFM imaging have been exploited in collaborative works investigating the mechanical properties of double-stranded RNA (JACS, 2013); the binding of peptides to DNA (ACSnano, 2013); and the characterisation of origami nano-structures to investigate translocation of molecules through nanopores. (ACSnano, 2013, Lab Chip, 2013).
