| DNA helicases are enzymes that catalyze DNA unwinding during replication, recombination, and repair. The bacteriophage T7 helicase (4A{dollar}spprime),{dollar} a hexameric helicase coupling dTTP hydrolysis to DNA unwinding, is a model for understanding helicase function. To examine the structure-function relationship and energy transduction mechanism, I characterized nine 4A{dollar}spprime{dollar} random mutant proteins that were selected for a helicase defect in vivo. I assayed the mutants for DNA unwinding activity, dTTPase activity, hexamer formation, and DNA binding. Based on these results, I divided the mutants into three groups. Group A mutants (E348K, D424N, and S496F), were defective in dTTPase activity and probably are located in the dTTP binding site. Group B mutants (R487C and G488D), were defective in DNA-stimulated dTTPase activity and probably are located in a switch region linking the dTTP binding and DNA binding sites. Group C mutants (G116D, A257T, S345F and G451E), were defective in coupling dTTP hydrolysis and DNA unwinding. Using these results, I predicted the three dimensional structure of the 4A{dollar}spprime{dollar} helicase domain by comparing it with the known structures of RecA, F{dollar}sb1{dollar}-ATPase, and Rep and PcrA helicases. The group C (uncoupled) mutants and one group A mutant, E348K, provided an opportunity to examine the mechanism of energy transduction. First, comparing the DNA unwinding and dTTPase rates, I found that the E348K protein had the unusual property of having a 4 to 6-fold improvement in DNA unwinding efficiency (number of bp unwound per dTTP hydrolysis) over the wild-type. Second, using transient state kinetic analysis, I found that the uncoupled mutants had a faster product (dTDP or P{dollar}rmsb{lcub}i{rcub}){dollar} release step than the wild-type. The product release step is therefore important for coupling. Third, comparing the dTTPase rates using DNA of different lengths, I found that some of the uncoupled mutants processively translocated along ssDNA. This shows that translocating along ssDNA is not sufficient for DNA unwinding. Finally, in a theoretical study, I argue that the binding-change mechanism of the F{dollar}sb1{dollar}-ATPase is a model for the alternating site mechanisms of helicases and many other motor proteins. |
