The ATP-activated hexameric helicase of bacteriophage T4 (gp41) forms a stable primosome with a single subunit of T4-coded primase (gp61).
We have examined the formation of the primosome subassembly of the bacteriophage T4-coded DNA replication (elongation) complex from its helicase, primase, and DNA components. Previously, we had shown that the T4 helicase (gp41) exists in solution in a stable monomer left and right arrow dimer equilibrium at physiological protein (and salt) concentrations and forms a hexamer upon activation by ATP (or GTP) binding (Dong, F., Gogol, E. P., and von Hippel, P. H.(1995) J. Biol. Chem. 270, 7462-7473). Here we report that the T4 primase (gp61) is a monomer in solution under the same conditions, and that the ATP-activated helicase binds to a single gp61 primase molecule on appropriate DNA templates to reconstitute a stable primosome. We show that: (i) the gp41 helicase alone does not form a stable complex with DNA templates, although this helicase by itself can carry out moderately processive ATP-driven translocation along single-stranded DNA (Young, M. C., Schultz, D. E., Ring, D., and von Hippel, P. H.(1994) J. Mol. Biol. 235, 1447-1458); (ii) the primase alone does form a stable complex with DNA; (iii) the helicase can bind to the primase-DNA complex in the presence of ATP or GTP to form a stable ternary complex; (iv) this complex consists of six helicase subunits and one primase subunit; and (v) the reconstituted primosome is stable for at least 10 to 20 min after NTP cleavage and dissociation of the hydrolysis products. These results strongly suggest that the functional T4 DNA replication primosome consists of an integrated 6:1 helicase-primase complex bound to DNA, and that the ATP-activated helicase hexamer remains intact throughout the processive DNA replication process.
Dong
F
F
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403-1229, USA.
von Hippel
P H
PH
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004789:Enzyme Activation; D006868:Hydrolysis; D010710:Phosphates / Q000378:metabolism; D013698:Templates, Genetic; D014764:Viral Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D010710:Phosphates; D014764:Viral Proteins; C063983:gene 41 protein, Enterobacteria phage T4; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA