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.Helicases are ubiquitous enzymes that play pivotal roles in diverse cellular activities (1). Reactions mediated by helicases require their ability to bind and move along a DNA or RNA strand. Such a fundamental feature is coupled to the binding of nucleoside triphosphates (NTPs) 2 and their hydrolysis. An underlying mechanism in helicase action is a change in contact of the protein with the nucleic acid strand depending on the state of the bound NTP (1). Binding and subsequent hydrolysis of NTP induce conformational shifts in the nucleic acid-interacting part of the helicase that enable the enzyme to move unidirectionally. The unidirectional movement enables the helicase to translocate on the strand to which it is bound, and upon encountering duplex DNA, its continued movement results in unwinding of the dsDNA. Consequently, despite the divergence in their structure and mechanism of action, all helicases require the binding and hydrolysis of NTP to fulfill these multiple roles (2). Accordingly, signature structural motifs of helicase for NTP binding, for example the Walker motifs A and B, are conserved throughout the helicase family (3). These motifs interact with the phosphate moiety of NTP and influence DNA binding upon hydrolysis of NTP. In the case of hexameric helicases such as the T7 DNA helicase, complexity of helicase action is increased because the oligomerization of the subunits is a prerequisite for all the steps described above. In helicases of this family, binding and hydrolysis of NTP oc...