Abstract
Homologous recombination is an important DNA repair pathway that plays key roles in maintaining genome stability. Escherichia coli RecA is an ATP-dependent DNA-binding protein that catalyzes the DNA strand exchange reactions in homologous recombination. RecA assembles into long helical filaments on single-stranded DNA, and these presynaptic complexes are responsible for locating and pairing with a homologous duplex DNA. Recent single molecule studies have provided new insights into RecA behavior, but the potential influence of ATP in the reactions remains poorly understood. Here we examine how ATP influences the ability of the RecA presynaptic complex to interact with homologous dsDNA. We demonstrate that over short time regimes, RecA presynaptic complexes sample heterologous dsDNA similarly in the presence of either ATP or ATPγS, suggesting that initial interactions do not depend on ATP hydrolysis. In addition, RecA stabilizes pairing intermediates in three-base steps, and stepping energetics is seemingly unaltered in the presence of ATP. However, the overall dissociation rate of these paired intermediates with ATP is ∼4-fold higher than with ATPγS. These experiments suggest that ATP plays an unanticipated role in promoting the turnover of captured duplex DNA intermediates as RecA attempts to align homologous sequences during the early stages of recombination.
Highlights
Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition [1,2,3,4]
Our work suggests that ATP hydrolysis may play an unanticipated role in the homology search process by promoting more rapid turnover of short tracts of microhomology from the presynaptic complex
ATP Hydrolysis and the Homology Search—Our work demonstrates that the reactions that take place during the early stages of duplex DNA interrogation and capture by the RecA presynaptic complex are qualitatively similar in the presence of ATP or the slowly hydrolyzed analog ATP␥S
Summary
Visualizing RecA Presynaptic Filaments with ssDNA Curtains—We used ssDNA curtains and total internal refection fluorescence microscopy to visualize RecA presynaptic complexes (Fig. 1A). The ssDNA substrate was generated using M13 as a template for rolling circle replication and anchored to a lipid bilayer within a microfluidic chamber through a biotinstreptavidin linkage and aligned along chromium barriers by application of hydrodynamic force, as previously described (24 –26). The ssDNA unravels when incubated with RPAeGFP, and the downstream ends of the RPA-ssDNA are anchored to exposed chromium pedestals through nonspecific adsorption. These assembly stages of the experiment rely upon the use of eukaryotic RPA because RPA does not extensively compact ssDNA, allowing preparation of double-tethered ssDNA curtains. Bacterial SSB causes extensive ssDNA compaction in our assays [27], and we have not yet
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
