Promiscuous Usage of Nucleotides by the DNA Helicase of Bacteriophage T7

Abstract

The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4) provides both helicase and primase activity at the replication fork. T7 DNA helicase preferentially utilizes dTTP to unwind duplex DNA in vitro but also hydrolyzes other nucleotides, some of which do not support helicase activity. Very little is known regarding the architecture of the nucleotide binding site in determining nucleotide specificity. Crystal structures of the T7 helicase domain with bound dATP or dTTP identified Arg-363 and Arg-504 as potential determinants of the specificity for dATP and dTTP. Arg-363 is in close proximity to the sugar of the bound dATP, whereas Arg-504 makes a hydrogen bridge with the base of bound dTTP. T7 helicase has a serine at position 319, whereas bacterial helicases that use rATP have a threonine in the comparable position. Therefore, in the present study we have examined the role of these residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity. Our results show that Arg-363 is responsible for dATP, dCTP, and dGTP hydrolysis, whereas Arg-504 and Ser-319 confer dTTP specificity. Helicase-R504A hydrolyzes dCTP far better than wild-type helicase, and the hydrolysis of dCTP fuels unwinding of DNA. Substitution of threonine for serine 319 reduces the rate of hydrolysis of dTTP without affecting the rate of dATP hydrolysis. We propose that different nucleotides bind to the nucleotide binding site of T7 helicase by an induced fit mechanism. We also present evidence that T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.Helicases are molecular machines that translocate unidirectionally along single-stranded nucleic acids using the energy derived from nucleotide hydrolysis (1-3). The gene 4 protein encoded by bacteriophage T7 consists of a helicase domain and a primase domain, located in the C-terminal and N-terminal halves of the protein, respectively (4). The T7 helicase functions as a hexamer and has been used as a model to study ring-shaped replicative helicases. In the presence of dTTP, T7 helicase binds to single-stranded DNA (ssDNA) 3 as a hexamer and translocates 5Ј to 3Ј along the DNA strand using the energy of hydrolysis of dTTP (5-7). T7 helicase hydrolyzes a variety of ribo and deoxyribonucleotides; however, dTTP hydrolysis is optimally coupled to DNA unwinding (5).Most hexameric helicases use rATP to fuel translocation and unwind DNA (3). T7 helicase does hydrolyze rATP but with a 20-fold higher K m as compared with dTTP (5, 8). It has been suggested that T7 helicase actually uses rATP in vivo where the concentration of rATP is 20-fold that of dTTP in the Escherichia coli cell (8). However, hydrolysis of rATP, even at optimal concentrations, is poorly coupled to translocation and unwinding of DNA (9). Other ribonucleotides (rCTP, rGTP, and rUTP) are either not hydrolyzed or the poor hydrolysis observed is not coupled to DNA unwinding (8). Furthermore, Patel et al. (10) found that the form of T7 helicase found in v...

Residues in the central β-hairpin of the DNA helicase of bacteriophage T7 are important in DNA unwinding

“…Phage complementation assay, ssDNA-dependent and independent dTTP hydrolysis assay, ssDNA-binding assay, dsDNA unwinding assay, protein oligomerization assay, and leading-strand DNA synthesis assay were performed as described in previously (36). Details of assay procedures can be found in the SI Text.…”

Coupling dTTP Hydrolysis with DNA Unwinding by the DNA Helicase of Bacteriophage T7

“…After incubation of the reactions up to 30 min, the reaction was stopped with EDTA at a final concentration of 25 mM and then the mixture was spotted onto DE81 filter paper. After washing 3 times with 0.3 M ammonium formate and 100% ethanol, incorporated [ 32 P]dTMP was measured in a liquid scintillating counter (28).…”

Direct role for the RNA polymerase domain of T7 primase in primer delivery

“…In the DNA unwinding assay, a radiolabeled minireplication fork (500 fmol) was incubated at 37°C for 5 min with the indicated gp4 in the presence of 1 mM dTTP. ssDNA formed as the result of unwinding was separated using a nondenaturing gel and the amount of ssDNA was determined (34). In RNA-primed DNA synthesis, reaction mixtures contain 10 nM M13 ssDNA, 0.3 mM all four dNTPs, 0.1 μCi of [α-32 P] dGTP, 100 nM T7 DNA polymerase (a 1∶1 complex of T7 gene 5 protein and E. coli thioredoxin), the indicated amounts of gp4, and either 0.1 mM of all NTP or the indicated amount of oligoribonucleotide.…”