טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRani Zananiri
SubjectEnsemble and Single Molecule Studies of DNA Unwinding by
RecBCD
DepartmentDepartment of Biology
Supervisors Professor Kaplan Ariel
Professor Henn Arnon
Full Thesis textFull thesis text - English Version


Abstract

RecBCD, responsible for the initiation of double strand break repair in bacteria, is a processive DNA helicase with an unwinding rate approaching ~1,600 bp•s-1. In addition, RecBCD possesses the ability to dislocate DNA binding proteins along its DNA track without pausing or slowing down. The mechanism enabling RecBCD to achieve such fast unwinding rates, while also generating the force required to cope with roadblocks, is not known. We employed a combination of equilibrium and time-resolved binding experiments, and ensemble and single molecule activity assays to uncover the molecular mechanism underlying RecBCD’s rapid ATP catalysis. We report the existence of auxiliary binding sites, where ATP binds with lower affinity and with distinct chemical interactions as compared to the known catalytic sites. The catalytic rate of RecBCD is reduced both by preventing and by strengthening ATP binding to these sites, suggesting that the dynamics of ATP at these sites modulates the enzyme’s rate. We propose a model by which RecBCD achieves its fast unwinding rate by utilizing the weaker binding sites to increase the flux of ATP to its catalytic sites. Next, we characterized RecBCD’s force generation mechanism and revealed that RecBCD is able to operate against a large range of forces reaching up to ~40pN without a significant effect on its velocity. Additionally, we characterized the force-dependence of the individual subunits’ translocation velocity, both when isolated and as part of the RecBCD complex. Interestingly, we show that RecD is only a weak helicase, but has the ability to translocate very fast. Thus, we propose a model by which, in the native complex, RecD does not play a role in destabilizing the fork, but translocates on the ssDNA formed after the RecC destabilizing “pin” motif. Lastly, we show that RecB unwinds the DNA using either a power-stroke model coinciding with hydrolysis or as a Brownian ratchet that is rectified by ATP hydrolysis. RecD, on the other hand, behaves like a Brownian ratchet that precedes ATP binding, and exhibits synergy shifting it towards the post translocation state when in complex with RecBCD.