| E. coli DNA helicase II (UvrD) has been shown to be required for DNA excision repair, methyl-directed mismatch repair and has some, yet undefined, role in DNA replication and recombination. However, little is known about the molecular mechanism for UvrD catalyzed DNA unwinding. Site-directed mutagenesis has been used to demonstrate the importance of conserved amino acid residues in the so-called "helicase motifs" (Brosh & Matson, 1996; George et al., 1994; Hall & Matson, 1997; Hall et al., 1998b). Many of these residues make contacts with the nucleotide or DNA substrate, but how these contacts translate into the coupling of nucleoside 5' triphosphate (NTP) hydrolysis to the disruption of hydrogen bonds holding the two strands, together is still not understood (for review see Lohman & Bjornson, (1996)). It has been my goal to begin to elucidate this mechanism for UvrD.;The role of the C-terminus of DNA helicase II, a region outside of the conserved helicase motifs, was investigated through the construction of three C-terminal truncation mutants: UvrDDelta107C, UvrDDelta102C, and UvrDDelta40C. The C-terminal region lacks sequence similarity with other helicases, and may function to tailor UvrD for its specific in vivo roles. Results from genetic complementation assays and biochemical assays suggest a role for a region new the C-terminus of UvrD in binding to single-stranded DNA. This region of the protein is essential for wild-type activity both in vitro and in vivo.;Interestingly, one of the C-terminal truncation mutants, UvrDDelta40C, was unable to self-associate and was primarily monomeric in solution. This was surprising since it has been assumed that helicases must be oligomeric to function. The UvrDDelta40C mutant, on the other hand, was as active as the wild-type enzyme in all biochemical and genetic assays examined. Therefore, I characterized the association state of wild-type UvrD and have demonstrated that while UvrD is capable of dimerization, UvrD is active as a monomer. The role of dimerization, if any, is not understood.;To gain additional insight into the structure and function of UvrD, I have initiated an effort to determine the structure of UvrD by X-ray crystallography. The purification of UvrD was improved and crystallization trials with UvrD, UvrD + ssDNA, UvrDDelta40C, and UvrDDelta40C + ssDNA were performed. Small crystals were produced but none large enough for diffraction studies. These results have provided a detailed description of the solubility characteristics of UvrD and UvrDDelta40C. Importantly, the solubility of UvrDDelta40C is improved over the wild-type protein.;Finally, I sought to relate our understanding of the unwinding mechanism of UvrD in vitro, to its biological role in vivo . Toward that end, I have been examining the interaction of UvrD with the mismatch repair protein, MutL. Previously, it had been shown that MutL dramatically stimulates the unwinding reaction catalyzed and physically interacts with UvrD. However the mechanism for this stimulation is not known. I have initiated experiments designed to elucidate the mechanism of this stimulation. The unwinding reaction by UvrD is considered to occur in three phases: loading and initiation, progressive unwinding, and completion. The data suggests that MutL loads UvrD onto the DNA, increasing both the rate of initiation, and the rate of progressive unwinding. |
