The initiation and progression of DNA unwinding by the bacteriophage T7 GP4 helicase: Kinetic studies, single molecule techniques, and computer modeling

Helicases are ubiquitous enzymes involved in many processes of DNA or RNA metabolism. The T7 helicase/primase is a replicative helicase providing ssDNA to the replication machinery at the replication fork, using free energy from dTTP hydrolysis. The interactions of T7 gp4 helicase/primase with DNA substrates conducive to DNA unwinding were investigated. The initiation complex was characterized to determine the requirement for ssDNA tails for in vitro initiation of DNA unwinding. Kinetic studies were carried out to determine the mechanism for the formation of the initiation complex. Finally, the kinetics of DNA unwinding provided insights into the mechanism of DNA unwinding after initiation. Functional studies of DNA unwinding showed that the optimal synthetic DNA substrate for unwinding contains a 5 ′-tail 35 bases in length and a 3′-tail 15 bases in length. The studies suggested that the formation of the initiation complex was rate-limiting but that this complex could be pre-formed in the absence of magnesium ions. Interaction with ssDNA in both strands was required during initiation and unwinding. Stopped-flow fluorescence studies suggested a mechanism for the formation of the initiation complex. Together with functional studies of dTTP hydrolysis and DNA unwinding, this confirmed a five-step mechanism for the formation of an enzymatically competent helicase-DNA complex. The rate-limiting step is the conversion of a stably bound inactive complex into an active complex. Single-molecule studies, namely atomic force microscopy, confirmed that a single, homohexameric helicase complex is capable of processively unwinding dsDNA. The mechanism of DNA unwinding was investigated by studying the kinetics of this process. The helicase translocates along ssDNA with a rate of about 80 s−1 at 18°C and unwinds DNA at a rate of about 40 bp/s. It appears that the same process that links the energy from the hydrolysis of dTTP to translocation along ssDNA also provides the force required for dsDNA unwinding. However, specific interactions with the DNA, beyond those required for translocation, seem to be necessary for DNA unwinding. A combination of kinetic biochemical, and single molecule studies are needed to understand the details of these processes.