Promiscuous Usage of Nucleotides by the DNA Helicase of Bacteriophage T7: DETERMINANTS OF NUCLEOTIDE SPECIFICITY

Ajit K Satapathy,Donald J Crampton,Benjamin B Beauchamp,Charles C Richardson

doi:10.1074/jbc.m900557200

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.

Highlights

Helicases are molecular machines that translocate unidirectionally along single-stranded nucleic acids using the energy derived from nucleotide hydrolysis [1,2,3]
We present evidence that T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding
A crystal structure of the T7 helicase domain bound to dATP reveals an interaction of the deoxyribose of dATP (Fig. 1) with Arg-363, whereas no interaction was observed in the structure with dTTP

Summary

Introduction

Helicases are molecular machines that translocate unidirectionally along single-stranded nucleic acids using the energy derived from nucleotide hydrolysis [1,2,3]. T7 helicase hydrolyzes a variety of ribo and deoxyribonucleotides; dTTP hydrolysis is optimally coupled to DNA unwinding [5]. Hydrolysis of rATP, even at optimal concentrations, is poorly coupled to translocation and unwinding of DNA [9]. The location of the nucleotide binding site at the subunit interface provides multiple interactions of residues with the bound NTP. His-465 (phosphate sensor), Glu-343 (catalytic base), and Asp424 (Walker motif B) interacts with the ␥-phosphate of the bound nucleotide in the same subunit [12, 17, 18]. The arginine finger and the phosphate sensor have been proposed to couple NTP hydrolysis to DNA unwinding. The conserved Lys-318 in Walker motif A interacts with the ␤-phosphate of the bound nucleotide and plays an important role in dTTP hydrolysis [21]. Considering the wealth of information on the above residues that are involved in the hydrolysis of dTTP and the coupling of VOLUME 284 NUMBER 21 MAY 22, 2009

Methods

Results

Conclusion