Helicases are motor proteins that use the free energy from NTP hydrolysis to translocate along and separate the strands of dsDNA/RNA. Defects in helicases have been shown to lead to genetic instability that manifests in diseases such as cancer and premature aging in humans. To understand the enzymatic mechanisms of helicases, we are studying two members of the hexameric class of helicases-the DNA helicase-primase from phage T7 (gp4A') and the transcription terminator Rho from E. coli. Studies in the previous grant period of NTP binding/ hydrolysis and DNA binding showed that the hexamer subunits act cooperatively, and based on these studies we proposed a 3-site sequential mechanism. In this mechanism, three catalytic sites hydrolyze NTP in a sequential manner promoting sequential DNA bind-release steps and directional movement of the helicase on the ssDNA. Based on single turnover DNA unwinding studies, in the previous grant period, we have proposed a """"""""DNA-exclusion"""""""" model. In this model, the helicase subunits surround only the lagging strand of the fork-DNA and exclude the leading strand from the central channel. We postulate that the helicase translocates unidirectionally on the lagging strand in the 5'-3' direction, but the mechanism of dsDNA unwinding is yet unknown. The studies in the next grant period will investigate the cooperative NTPase catalysis and the DNA unwinding mechanisms.
The specific aims of these studies are as follows. 1) To determine the mechanism of NT? hydrolysis catalyzed by T7 gp4A and E. coli Rho hexamers bound to DNA/RNA, using single-turnover and presteady-state NTPase kinetics. 2) To characterize selected """"""""uncoupled"""""""" mutants of T7 gp4A' to gain insights into the enzymatic steps that are critical in coupling the NTPase activity to unwinding. 3) To use protein-DNA photo cross-linking studies to characterize the interactions of T7 gp4A' hexamer with ssDNA and fork-DNA, and map the protein regions that interact with specific parts of the substrate DNAs. 4) To characterize the unwinding reaction by determining the step-size, stepping rate, and the processivity of unwinding using single-turnover experiments, first in the absence and then in the presence of T7 DNA polymerase.
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