|Ph.D Student||Vera Gaydar|
|Subject||Unique Enzymatic Adaptation for the Super Fast RecBCD DNA|
|Department||Department of Biology||Supervisor||Professor Henn Arnon|
|Full Thesis text|
RecBCD is a highly processive bacterial DNA helicase important for double-strand break repair pathway. RecBCD possesses multi-enzymatic activities specifically designed for its function, including an extremely fast unwinding rate of ~ 1,600 bp・s-1, consuming a very high ATP turnover. To do so, it had reached near catalytic perfection being an order of magnitude faster than any other measured helicases unwinding rates. Yet, poorly understood are the nucleotide binding linkage, the biochemical transitions of RecBCD and how the high rate of ATP turnover is mechanistically achieved by RecBCD motor. In this work we present thermodynamic and kinetic analysis of nucleotides’ binding to RecBCD and RecBCD・DNA complexes, and specifically how salt modulates DNA unwinding velocities. Our results reveal new and unique enzymatic adaptations by RecBCD to achieve rapid ATPase cycling time. We revealed new weak nucleotides binding sites in addition to the known canonical strong binding sites of RecBCD. These weak nucleotides binding sites are evident in both our thermodynamics and transient kinetic measurements and are essential to RecBCD rapid catalysis. In addition, we confirm the results by RecBCD mapping analysis. We revealed many putative weak nucleotides binding sites especially near strong catalytic binding sites. Furthermore, we performed rigorous Gibbs free energy analysis and revealed that RecBCD reaction mechanism is driven by minimizing conformational changes along its reaction coordinates as determined by small changes in the free energy of the different ligated states of RecBCD. We substantiate these results by SAXS measurements comparing between RecBCD to RecBCD・hpDNA・ADP complex. Corollary, our work suggests a mechanism by which RecBCD achieves rapid unwinding rates by weak nucleotide binding sites that act as a ‘buffer’ to increase the local concentration of accessible ATP during rapid unwinding and by minimizing the conformational changes along the reaction coordinate during unwinding.