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

Kristen Moore Picha,Smita S Patel

doi:10.1074/jbc.273.42.27315

Abstract

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 kcat for the dTTPase activity was 200-300-fold lower in the absence of MgCl2, but the Km was only slightly affected. Direct dTTP binding experiments showed that the Kd 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+. In 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.

Highlights

Helicases are motor proteins that translocate on DNA and catalyze separation of the double-stranded DNA into singlestranded DNAs
To investigate the role of Mg2ϩ and dTTP in hexamer formation, hydrolysis, and DNA binding by T7 4AЈ protein, we have characterized the activities of 4AЈ protein both in the presence and absence of Mg2ϩ
As the concentration of EDTA was increased from 0.5 ␮M to 20 mM, the rate of dTTP hydrolysis decreased and was saturated by 0.5–1 mM EDTA

Summary

EXPERIMENTAL PROCEDURES

Nucleotides and Other Reagents—[␣-32P]dTTP was purchased from Amersham Pharmacia Biotech, and its purity was checked and corrected for in all of the experiments. dTTP was purchased from Sigma. The NC and DEAE membranes were washed with 0.5 M NaOH for 10 min, rinsed with double-distilled water, and equilibrated with the membrane wash buffer (50 mM Tris-Cl (pH 7.5), 5 mM NaCl, and either 10 mM MgCl2 or 2 mM EDTA) for at least 1 h at room temperature. The assays were performed at constant DNA (1 ␮M) and increasing 4AЈ (0 –20 ␮M) concentration in buffer containing 50 mM Tris-Cl (pH 7.5), 40 mM NaCl, 10% glycerol, and in the presence of 10 mM MgCl2 or 0.5 mM EDTA. After the samples were filtered, radioactivity on both NC and DEAE filters was quantitated using a PhosphorImager (Molecular Dynamics)

RESULTS

No DNA

DISCUSSION