Characterizing the auxiliarynucleotide binding sites in RecBCDDNA helicase, towards deciphering their functional role
Abstract:
RecBCD is aDNA helicase-nuclease, powering the initiation of dsDNA break repair. It is ahighly processive fast helicase, with unwinding rates approaching 1,600 bp/s.Yet, there is no underlying biophysical model to understand this fast-unwindingvelocity. To unravel RecBCD’smechanochemical basis for its robust performance on DNA, we have employedquantitative biophysical studies utilizing rapid kinetics, thermodynamics,cross-linking mass spectrometry (CLMS) and in-vivosurvival assays. Previous work in our lab had demonstrated the existence ofauxiliary binding sites in RecBCD, specifically in the RecCsubunit, where ATP binds with lower affinity and with distinct chemicalinteractions as compared to the known catalytic sites. According to our model,at intermediate ATP concentrations, RecBCD achieves its fast-unwinding rate byutilizing the auxiliary binding sites to increase the flux of ATP to itscatalytic sites. The number of nucleotide-binding sites determined byequilibrium dialysis has been estimated to be at least four. While RecB and RecD,each have one known structurally and chemically well-defined nucleotide-bindingsite, the additional two (or more) auxiliary nucleotide-binding sites remainedunknown. Our current study strongly supports that they are likely to be locatedin RecC. Inthis work, we have attempted to abolish the nucleotide-binding sites in RecC byusing a multi-step approach. A list of possible nucleotide-binding sites wasobtained by using a combination of CLMS and molecular docking studies. Based onthis, several mutations in RecC were carefully designed and successfullycloned and purified to produce a variety of RecBCmutDs. RecBCmutD proteins have an activated DNA ATPaseactivity, with a higher KM,ATP.Furthermore, equilibrium nucleotide binding, DNA unwinding, and invivosurvival assays suggest that the RecBCmutDs donot display the same biochemical behavior as the WT RecBCD. Inaddition, despite exhibiting rapid transition, as suggested by the rate ofunwinding performed by RecBCD, wecan still observe a stepping mechanism essential to translocate along its DNAtracks. Our work sheds light on a novel and fundamental enzymatic mechanismexhibited by a molecular motor.
Beyond the mechanism of RecBCD andhow it is evolved for its cellular function, the question of why such an enzymeto repair dsDNA break in humans did not evolve, continues to be pertinent.
