Allosteric Effects Of Recb Nuclease Domain On Recbcd-Dna Interactions

E. coli RecBCD is crucial in initiating repair of double stranded (ds) DNA breaks. It is a heterotrimeric helicase and nuclease complex possessing two ATPase motors, RecB and RecD, and a regulatory subunit without ATPase activity, RecC. The RecB subunit contains a 30kDa nuclease domain (RecBNuc) that, according to published structural data, is situated over 60Å away from the site of dsDNA binding. Surprisingly, we have shown that deletion of RecBNuc to form RecBΔNucCD affects its dsDNA unwinding properties. The mechanism by which RecBNuc influences RecBCD dsDNA unwinding is unclear. To probe this further, we examined how deletion of RecBNuc affects the thermodynamics of RecBCD binding to dsDNA ends using isothermal titration calorimetry (ITC) and fluorescence titrations. RecBCD can melt 6 base pairs upon binding a blunt ended DNA in an ATP-independent, but Mg2+-dependent reaction. We observe an unfavorable ΔH for binding of both RecBCD and RecBΔNucCD to blunt ended DNA, likely reflecting the cost of melting the 6 base pairs. RecBΔNucCD binds to dsDNA ends with much higher affinity than RecBCD, which is driven primarily by a more favorable ΔS°. Previous studies showed that RecBCD has optimal binding affinity for a dsDNA end possessing 3’dT6 and 5’dT10 ssDNA tails. Here, we present thermodynamic data (ΔG°, ΔH and TΔS°) showing a continued increase in favorable ΔH for binding dsDNA ends possessing ssDNA tails longer than 3’dT6 and 5’dT10. However, these more favorable ΔH values are compensated by unfavorable TΔS° values. This suggests that RecBCD makes contacts with dsDNA ends possessing ssDNA tails longer than 3’dT6 and 5’dT10, which was unexpected based on previous data. This ssDNA tail length effect differs for RecBΔNucCD. These data suggest that RecBNuc regulates RecBCD dsDNA interactions through long-range allosteric effects. (Supported by NIH grant GM045948.)