Heterogeneity in E. coli RecBCD Helicase-DNA Binding and Base Pair Melting |
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| Affiliation: | 1. The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada;2. Phialogics GmbH, Bettinastr. 30, 60325 Frankfurt am Main, Germany;3. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada;4. Lunenfeld‐Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada;1. Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA;2. Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA;3. Institute of Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen 518055, China;4. Institute of Metabolism and Systems Research, University of Birmingham, B15 2TT Birmingham, UK;5. Centre of Membrane Proteins and Receptors, Universities of Birmingham and Nottingham, B15 2TT Birmingham, UK;6. Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA;7. Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093, USA;1. Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, United States;2. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z4, Canada;1. The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA;2. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA;3. Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA;1. School of Biosciences, University of Kent, Canterbury, United Kingdom;2. Department of Biology & Biochemistry, University of Bath, Bath, United Kingdom |
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| Abstract: | E. coli RecBCD, a helicase/nuclease involved in double stranded (ds) DNA break repair, binds to a dsDNA end and melts out several DNA base pairs (bp) using only its binding free energy. We examined RecBCD-DNA initiation complexes using thermodynamic and structural approaches. Measurements of enthalpy changes for RecBCD binding to DNA ends possessing pre-melted ssDNA tails of increasing length suggest that RecBCD interacts with ssDNA as long as 17–18 nucleotides and can melt at least 10–11 bp upon binding a blunt DNA end. Cryo-EM structures of RecBCD alone and in complex with a blunt-ended dsDNA show significant conformational heterogeneities associated with the RecB nuclease domain (RecBNuc) and the RecD subunit. In the absence of DNA, 56% of RecBCD molecules show no density for the RecB nuclease domain, RecBNuc, and all RecBCD molecules show only partial density for RecD. DNA binding reduces these conformational heterogeneities, with 63% of the molecules showing density for both RecD and RecBNuc. This suggests that the RecBNuc domain is dynamic and influenced by DNA binding. The major RecBCD-DNA structural class in which RecBNuc is docked onto RecC shows melting of at least 11 bp from a blunt DNA end, much larger than previously observed. A second structural class in which RecBNuc is not docked shows only four bp melted suggesting that RecBCD complexes transition between states with different extents of DNA melting and that the extent of melting regulates initiation of helicase activity. |
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| Keywords: | DNA recombination SF1 helicase thermodynamics Cryo-EM |
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