Bacteriophage T7 DNA helicase binds dTTP, forms hexamers, and binds DNA in the absence of Mg2+ -: The presence of dTTP is sufficient for hexamer formation and DNA binding

The role of Mg2+ in dTTP hydrolysis, dTTP binding, hexamer formation, and DNA binding was studied in bacteriophage T7 DNA helicase (4A' protein). The steady state k(cat) for the dTTPase activity was 200-300-fold lower in the absence of MgCl2, but the K-m was only slightly affected, Direct dTTP binding experiments showed that the K-d of dTTP was unaffected, but the stoichiometry of dTTP binding was different in the absence of Mg2+. Two dTTPs were found to bind tightly in the absence of Mg2+ in contrast to three to four in the presence of Mg2+. I, the presence of DNA there was little difference in the stoichiometry of dTTP binding to 4A', These results indicate that Mg2+ is not necessary for dTTP binding, but Mg2+ is required for optimal hydrolysis of dTTP, Gel filtration of 4A' in the presence of dTTP without Mg2+ showed that Mg2+ was not necessary, and dTTP was sufficient for hexamer formation. The hexamers formed in the presence of dTTP without Mg2+ were capable of binding single-stranded DNA, However, the 4A' hexamers formed in the presence of dTDP with or without Mg2+ did not bind DNA, indicating that hexamer formation itself is not sufficient for DNA binding. The hexamers need to be in the correct conformation, in this case in the dTTP-bound state, to interact with the DNA. Thus, the gamma-phosphate of dTTP plays an important role in causing a conformational change in the protein that leads to stable interactions of 4A' with the DNA.