Stoichiometry and DNA Unwinding by the Bacteriophage T4 41:59 Helicase

Kevin D Raney,Theodore E Carver,Stephen J Benkovic

doi:10.1074/jbc.271.24.14074

Kevin D Raney, Theodore E Carver + Show 1 more

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https://doi.org/10.1074/jbc.271.24.14074

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Abstract

The bacteriophage T4 41 protein is a replicative helicase that forms a hexamer in the presence of ATP and associates with the T4 59 protein. The stoichiometry of the 41:59 helicase complex and its mechanism for DNA unwinding have been investigated using steady-state and single-turnover kinetics. A partial duplex DNA fork containing two regions of single-stranded DNA (ssDNA) of 30 nucleotides each, and 30 base pairs served as the substrate. 59 was found to increase the steady-state unwinding rate of the substrate by 200-fold over the rate of 41 alone. Maximum unwinding occurred when 59 and 41 were equimolar, revealing a 1:1 stoichiometry for the complex. Varying 41 while holding 59 constant resulted in sigmoidal kinetics suggesting strong cooperativity for formation of the 41 hexamer and providing a lower limit for hexamer assembly of 65 nM. Substrates were prepared that contained a biotin-streptavidin block in either the leading or lagging strand of the duplex region of the substrate. The first order rate constant for unwinding was reduced only when the block was placed in the lagging strand of the DNA fork, indicating that the helicase interacts primarily with the lagging DNA strand.

Highlights

Bacteriophage T4 DNA replication is facilitated by the phage-encoded 41 helicase. 41 is a single-stranded DNA(ssDNA)1 stimulated nucleotide triphosphatase with high affinity for ATP and GTP (Morrical et al, 1995; Young et al, 1994; Liu and Alberts, 1981)
Interaction of 41 with the T4 61 protein makes up the T4 primosome that is responsible for unwinding dsDNA for leading strand DNA synthesis as well as priming of the lagging strand for lagging strand DNA synthesis (Hinton and Nossal, 1987; Liu and Alberts, 1981; Burke et al, 1985). 41 assembles into a hexamer upon binding ATP or GTP, and this oligomer is believed to be the active form of the enzyme (Dong et al, 1995)
Development of a Rapid Kinetic Assay for Measuring DNA Unwinding—A DNA unwinding assay using rapid kinetics was developed for measuring helicase activity on oligonucleotide substrates (Fig. 1)

Summary

Introduction

Bacteriophage T4 DNA replication is facilitated by the phage-encoded 41 helicase. 41 is a single-stranded DNA(ssDNA) stimulated nucleotide triphosphatase with high affinity for ATP and GTP (Morrical et al, 1995; Young et al, 1994; Liu and Alberts, 1981). 41 is a single-stranded DNA(ssDNA) stimulated nucleotide triphosphatase with high affinity for ATP and GTP (Morrical et al, 1995; Young et al, 1994; Liu and Alberts, 1981). In the presence of 59, the activity of 41 as an ATPase and a helicase is restored, suggesting that 59 enables 41 to load onto ssDNA that is coated with 32 (Barry and Alberts, 1994; Morrical et al, 1994; Yonesaki, 1994; Tarumi and Yonesaki, 1995). Other helicases require an accessory protein to attain high levels of activity. The DNA B helicase has been shown to exist as hexamer (Reha-Krantz and Hurwitz, 1978; Bujalowski et al, 1994) as has RuvB (Stasiak et al, 1994), and there may be a similar mechanism of action for these helicase enzymes and their respective accessory proteins

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