Molecular Basis for Recognition of Nucleoside Triphosphate by Gene 4 Helicase of Bacteriophage T7

Seung-Joo Lee,Charles C Richardson

doi:10.1074/jbc.m110.156067

Abstract

The translocation of DNA helicases on single-stranded DNA and the unwinding of double-stranded DNA are fueled by the hydrolysis of nucleoside triphosphates (NTP). Although most helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To identify the structural requirements of the NTP, we determined the efficiency of DNA unwinding by T7 helicase using a variety of NTPs and their analogs. The 5-methyl group of thymine was critical for the efficient unwinding of DNA, although the presence of a 3'-ribosyl hydroxyl group partially overcame this requirement. The NTP-binding pocket of the protein was examined by randomly substituting amino acids for several amino acid residues (Thr-320, Arg-504, Tyr-535, and Leu-542) that the crystal structure suggests interact with the nucleotide. Although positions 320 and 542 required aliphatic residues of the appropriate size, an aromatic side chain was necessary at position 535 to stabilize NTP for efficient unwinding. A basic side chain of residue 504 was essential to interact with the 4-carbonyl of the thymine base of dTTP. Replacement of this residue with a small aliphatic residue allowed the accommodation of other NTPs, resulting in the preferential use of dATP and the use of dCTP, a nucleotide not normally used. Results from this study suggest that the NTP must be stabilized by specific interactions within the NTP-binding site of the protein to achieve efficient hydrolysis. These interactions dictate NTP specificity.

Highlights

Case to translocate on the strand to which it is bound, and upon encountering duplex DNA, its continued movement results in unwinding of the dsDNA
A recent structural study of an AAAϩ hexameric helicase demonstrated that a polypeptide hairpin of the helicase makes different contacts with the ssDNA depending on the hydrolysis state of the nucleoside triphosphates (NTP) (6)
We measured the ability of various NTPs to support activity of T7 DNA helicase to unwind DNA (Fig. 2)

Summary

Introduction

Case to translocate on the strand to which it is bound, and upon encountering duplex DNA, its continued movement results in unwinding of the dsDNA. The primase benefits from the relatively tight binding of the helicase to DNA, and its location in the N-terminal half of the protein places it in the proper position to survey the DNA exiting from the translocating helicase for primase recognition sites (10) Another feature unique to the T7 gene 4 helicase is its preference for dTTP, whereas most other helicases prefer ATP, the most abundant NTP in the cell (11), in the unwinding reaction (12–15). Preferential use of dTTP by T7 gene 4 protein was noticed initially in studies on strand displacement DNA synthesis mediated by T7 DNA polymerase and T7 DNA helicase (18) In this reaction, the helicase unwinds the duplex DNA to generate ssDNA template for the DNA polymerase. The residues mentioned above are located in proximity to phosphate groups of the NTP, structural studies demonstrate that the base of the NTP is stacked between the side chains of two residues, Tyr-535 and Arg-504 (9), implicating a role for these residues in NTP binding

Methods

Results

Conclusion