The Phage T4-coded DNA Replication Helicase (gp41) Forms a Hexamer upon Activation by Nucleoside Triphosphate (∗)

• Published in: The Journal of biological chemistry Vol. 270; no. 13; pp. 7462 - 7473
• Main Authors: Dong, Feng, Gogol, Edward P., von Hippel, Peter H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31-03-1995; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacology; Animals; Bacteriophage T4 - enzymology; Base Sequence; DNA - biosynthesis; DNA - chemistry; DNA Helicases - chemistry; DNA Helicases - isolation & purification; DNA Helicases - metabolism; DNA Replication; DNA, Viral - biosynthesis; Enzyme Activation; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; Guanosine Triphosphate - metabolism; Guanosine Triphosphate - pharmacology; Macromolecular Substances; Mice; Molecular Sequence Data; Nucleic Acid Conformation; phage T4; Ribonucleotides - metabolism; Ribonucleotides - pharmacology; Substrate Specificity; Viral Proteins - chemistry; Viral Proteins - isolation & purification; Viral Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.13.7462

Sedimentation and high performance liquid chromatography studies show that the functional DNA replication helicase of bacteriophage T4 (gp41) exists primarily as a dimer at physiological protein concentrations, assembling from gp41 monomers with an association constant of ∼106M−1. Cryoelectron microscopy, analytical ultracentrifugation, and protein-protein cross-linking studies demonstrate that the binding of ATP or GTP drives the assembly of these dimers into monodisperse hexameric complexes, which redissociate following depletion of the purine nucleotide triphosphatase (PuTP) substrates by the DNA-stimulated PuTPase activity of the helicase. The hexameric state of gp41 can be stabilized for detailed study by the addition of the nonhydrolyzable PuTP analogs ATPγS and GTPγS and is not significantly affected by the presence of ADP, GDP, or single-stranded or forked DNA template constructs, although some structural details of the hexameric complex may be altered by DNA binding. Our results also indicate that the active gp41 helicase exists as a hexagonal trimer of asymmetric dimers, and that the hexamer is probably characterized by D3 symmetry. The assembly pathway of the gp41 helicase has been analyzed, and its structure and properties compared with those of other helicases involved in a variety of cellular processes. Functional implications of such structural organization are also considered.