Interactions of Bacteriophage T4-coded Primase (gp61) with the T4 Replication Helicase (gp41) and DNA in Primosome Formation

Abstract

One primase (gp61) and six helicase (gp41) subunits interact to form the bacteriophage T4-coded primosome at the DNA replication fork. In order to map some of the detailed interactions of the primase within the primosome, we have constructed and characterized variants of the gp61 primase that carry kinase tags at either the N or the C terminus of the polypeptide chain. These tagged gp61 constructs have been probed using several analytical methods. Proteolytic digestion and protein kinase protection experiments show that specific interactions with single-stranded DNA and the T4 helicase hexamer significantly protect both the N-and the Cterminal regions of the T4 primase polypeptide chain against modification by these procedures and that this protection becomes more pronounced when the primase is assembled within the complete ternary primosome complex. Additional discrete sites of both protection and apparent hypersensitivity along the gp61 polypeptide chain have also been mapped by proteolytic footprinting reactions for the binary helicase-primase complex and in the three component primosome. These studies provide a detailed map of a number of gp61 contact positions within the primosome and reveal interactions that may be important in the structure and function of this central component of the T4 DNA replication complex.Most known DNA replication complexes, including the bacteriophage T4-coded system, are organized into subassemblies of proteins that perform equivalent functions within the moving replication fork. Since the T4 complex is probably the simplest replication system that includes separate processivity clamp and clamp loader and primosome subassemblies, the T4 system has often served as a useful paradigm to elucidate and model some of the mechanistically important interactions that also occur within the replication complexes of higher organisms.The central replication machinery of bacteriophage T4 consists of seven phage-encoded proteins that work together with the DNA of the replication fork to form the minimal complex that permits efficient leading and lagging DNA synthesis and replication fork movement (1). These protein components are organized into three functional subassemblies within the replication complex. These are (i) the DNA-dependent DNA replication polymerase of T4 (gp43), which contains both synthesis and exonuclease (3Ј 3 5Ј) editing domains and works together with the T4-coded single-stranded DNA binding protein (gp32) to catalyze DNA synthesis on both strands of the replication fork; (ii) the T4-coded polymerase accessory proteins (gp44/62 and gp45), which form the processivity clamp (gp45) and ATPdependent clamp-loading (gp44/62) machinery of the T4 system; and (iii) the T4 coded helicase (gp41) and primase (gp61) components, which interact to form a primosome subassembly that contains an ATP-dependent helicase (gp41) that "opens" the template DNA ahead of the replication fork and a primase (gp61) that catalyzes the template-directed polymerization of the RNA primers that initiate th...

Assembly and subunit stoichiometry of the functional helicase-primase (primosome) complex of bacteriophage T4

“…This is compatible with a ssDNA binding site size for the T4 helicase of approximately 20 nts (18) and is also consistent with the binding site size that has been demonstrated for the closely related hexameric dnaB helicase of E. coli (19). (ii) The primase subunit binds to two ssDNA-bound gp41 subunits at the replication fork (20), bringing it into effective contact with the NTP ligand that lies between these subunits. This primase binding proximity could then specifically "activate" the hydrolysis of the NTP located at this position, while not perturbing the NTP ligands that lie between and stabilize other gp41 subunit interfaces and are not directly adjacent to the primase subunit.…”

Interaction of adjacent primase domains within the hexameric gene 4 helicase-primase of bacteriophage T7

“…Third, translocation of gene 4 protein by the helicase domain transports the primase domain in its search for primase recognition sites. Such a dependency of primase activity on helicase activity also is observed in both the E. coli or T4 phage replication systems in which the two proteins are encoded by separate genes but form a stable complex (6,26,27). Finally, each primase subunit in the hexameric gene 4 protein is in close proximity to neighboring subunits.…”

“…Therefore, expression of the 56-kDa protein can be an economic strategy for the phage to save production of unnecessary zinc motifs in the 63-kDa subunits in the hexamer. Indeed, it is suggested that less than three molecules of primase form a stable complex with one hexamer of helicase from T4 phage and Bacillus stearothermophilus (6,27).…”

Heterohexamer of 56- and 63-kDa Gene 4 Helicase-Primase of Bacteriophage T7 in DNA Replication

“…The binding of the T4 primase to its cognate helicase stimulates helicase activity (47). It has been suggested that three or less primases form a stable complex with a helicase hexamer in T4 phage and Bacillus stearothermophilus (48,49). Bacteriophage T7 expresses equal molar amounts of the 63-and 56-kDa gp4.…”