Kinetic Pathway of dTTP Hydrolysis by Hexameric T7 Helicase-Primase in the Absence of DNA

Abstract

Bacteriophage T7 gp4A protein is a hexameric helicase-primase protein that separates the strands of a duplex DNA in a reaction coupled to dTTP hydrolysis. Here we reexamine in more detail the kinetic mechanism of dTTP hydrolysis by a preassembled T7 helicase hexamer in the absence of DNA. Pre-steady state dTTP hydrolysis kinetics showed a distinct burst whose amplitude indicated that a preformed hexamer of T7 helicase hydrolyzes on an average one dTTP per hexamer. The presteady state chase-time experiments provided evidence for sequential hydrolysis of two dTTPs. The medium [ 18 O]P i exchange experiments failed to detect dTTP synthesis, indicating that the less than six-site hydrolysis observed is not due to reversible dTTP hydrolysis on the helicase active site. The P i -release rate was measured directly using a stopped-flow fluorescence assay, and it was found that the rate of dTTP hydrolysis on the helicase active site is eight times faster than the P i -release rate, which in turn is three times faster than the dTDP release rate. Thus, the rate-limiting step in the pathway of helicase-catalyzed deoxythymidine triphosphatase Helicases are motor proteins that translocate along nucleic acids using the energy of NTP 1 hydrolysis. This activity facilitates many nucleic acid metabolic processes including those that require the separation of double-stranded DNA into their component single strands (1-3). A class of helicases assembles into ring-shaped hexamers, and helicases belonging to this class have been found to function in genome replication and recombination that require processive action of the helicase (1). The assembly of helicase subunits into hexamers is generally induced by NTP binding, and the hexamer is also stabilized by DNA (1, 4 -6). Bacteriophage T7 gp4 protein is one of the better-studied ring helicases whose function is to facilitate T7 genome replication and recombination. T7 gp4 proteins gp4AЈ and gp4B assemble into rings in the presence of nucleotide triphosphate, preferably dTTP or its non-hydrolyzable analog dTMP-PCP. The hexamer has a high affinity for ssDNA that binds within the central channel of the ring. It has been proposed that the helicase passes only one strand of the doublestranded DNA through the central channel and excludes the complementary strand from the central channel as it translocates and separates the strands of the double-stranded DNA (7-10).The translocation and strand separation activity of T7 helicase is fueled by dTTP hydrolysis. The mechanism by which dTTP hydrolysis is coupled to translocation and strand separation is not known. Our previous studies indicate that the dTTPase mechanism of gp4AЈ shows striking similarity to the binding change mechanism of the F 1 -ATPase protein (11) despite there being no amino acid sequence homology between them. These studies showed that two-three dTTPs bound tightly but did not get hydrolyzed at the steady state rate, and these were referred to as the noncatalytic sites. Two additional dTTPs, referred to as catalytic sites, we...

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“…Phosphate (P i ) release was assayed in real-time using a fluorescent probe (MDCC) attached to E. coli PBP as described previously (39)(40)(41). Change in fluorescence of MDCC-PBP upon P i binding was monitored using an excitation wavelength of 425 nm and emission above 450 nm (cutoff filter, Corion LL-450 F).…”

“…A coupled enzyme reaction (Mop) containing 200 µM 7-methyl guanosine and 0.01 U/mL purine nucleoside phosphorylase was used in all reactions to sequester contaminant P i as ribose-1-phosphate, because MDCC-PBP is sensitive to micromolar concentrations of P i . A P i calibration curve relating the PBP-MDCC fluorescence signal to P i concentration was generated prior to each experiment, as described previously (39). All reactions and syringes were "mopped" for at least 45 min before each experiment.…”

Role of a Conserved Glutamate Residue in the Escherichia coli SecA ATPase Mechanism

“…A coupled enzyme reaction (Mop) containing 200 M 7-methylguanosine and 0.03 units/ml purine nucleoside phosphorylase was used in all reactions to sequester contaminant P i as ribose 1-phosphate, because MDCC-PBP is sensitive to micromolar concentrations of P i . A P i calibration curve relating the PBP-MDCC fluorescence signal to P i concentration was generated prior to each experiment (31). All reactions and syringes were "mopped" for at least 45 min before each experiment.…”

“…Phosphate (P i ) release was assayed in real-time using fluorescent MDCC-labeled E. coli PBP as described previously (31,34,35). Changes in MDCC-PBP fluorescence upon P i binding were monitored by excitation at 425 nm and monitoring emission above 450 nm (cut-off filter; Corion LL-450 F).…”

“…Pro-OmpA (28) and urea-treated inverted membrane vesicles (29) were prepared as described. Phosphate-binding protein (PBP) was purified and labeled with 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) as described (30,31 P]ADP was prepared as described previously (32), and non-radioactive nucleotides were purchased from Sigma. Polyethyleneimine cellulose-F TLC plates were purchased from EM Science and nitrocellulose membranes from Schleicher and Schuell.…”