The Oligomeric T4 Primase Is the Functional Form duringReplication

Abstract

Replisome DNA primases are responsible for the synthesis of short RNA primers required for the initiation of repetitive Okazaki fragment synthesis on the lagging strand during DNA replication. In bacteriophage T4, the primase (gp61) interacts with the helicase (gp41) to form the primosome complex, an interaction that greatly stimulates the priming activity of gp61. Because gp41 is hexameric, a question arises as to whether gp61 also forms a hexameric structure during replication. Several results from this study support such a structure. Titration of the primase/single-stranded DNA binding followed by fluorescence anisotropy implicated a 6:1 stoichiometry. The observed rate constant, k cat , for priming was found to increase with the primase concentration, implicating an oligomeric form of the primase as the major functional species. The generation of hetero-oligomeric populations of the hexameric primase by controlled mixing of wild type and an inactive mutant primase confirmed the oligomeric nature of the most active primase form. Mutant primases defective in either the N-or C-terminal domains and catalytically inactive could be mixed to create oligomeric primases with restored catalytic activity suggesting an active site shared between subunits. Collectively, these results provide strong evidence for the functional oligomerization of gp61. The potential roles of gp61 oligomerization during lagging strand synthesis are discussed.Primases play an indispensable role in DNA replication by synthesizing the RNA primers needed for initiation of de novo DNA synthesis (1). In eukaryotic cells, both leading and lagging strand syntheses are primed by the polymerase ␣-primase complex (2). In bacteriophage T4, the leading strand primer is synthesized by the Escherichia coli RNA polymerase during the early phase of phage DNA replication, or provided through strand invasion via homologous recombination in the late phase of replication (3). T4 gp61 1 primase then makes the short pentaribonucleotide primers required on the lagging strand to initiate repetitive Okazaki fragment synthesis (4).Gp61 primase recognizes two trinucleotide sequences in vitro, 5Ј-GTT-3Ј and 5Ј-GCT-3Ј, but uses only the former sequence in vivo as the priming site (5, 6). Although gp61 alone is able to synthesize primers on a single-stranded DNA (ssDNA) substrate, its maximum activity is achieved only through interaction with the helicase (gp41) to form the primosome complex (6, 7). Several other T4 proteins, including the helicase accessory protein (gp59) and the single-stranded DNA-binding protein (gp32), also modulate primase activity presumably by either facilitating the formation of the primosome or directly interacting with it (7).Among all the T4 replisome proteins, the helicase exhibits the greatest stimulatory effect on the priming activity of the primase. T4 helicase, like T7 and E. coli DnaB helicases (8, 9), acts as a hexamer and forms a ring structure encircling the lagging strand (10, 11). Movement of this hexameric ring is driven by ATP hy...

RNA Primer Handoff in Bacteriophage T4 DNA Replication

“…been shown to inhibit the activity of the entire primase oligomer (38). The results of this experiment indicate that the primase protein is much less processive in the presence of gp32-A than wt-gp32 (Table 1 and Fig.…”

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

“…In early work gel shift and protein-protein chemical cross-linking methodologies had shown that the gp61 primase subunit binds to the gp41 helicase hexamer at an approximately 1∶6 subunit stoichiometry (5,7). In contrast, Benkovic and coworkers have interpreted more recent and indirect fluorescence anisotropy and scanning calorimetry experiments (10)(11)(12) to argue that the primase subunits may interact with gp41 in an approximately 6∶6 subunit stoichiometry. This difference is important because in a primosome containing one primase and six helicase subunits the primase must be shared, which makes very different functional and mechanistic demands on both helicase and primase mechanisms than does a 6∶6 ratio, as is found in the hexameric helicase of bacteriophage T7, where every helicase subunit carries its own primase domain (13).…”

Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity

“…This assay makes possible the separation of binding and catalytic parameters and the study of real-time kinetics. It is based on steady-state fluorescence anisotropy (r), which is sensitive to rotational diffusion, and thus suitable for studies aiming to identify structural modifications leading to a significant change in molecular size (12)(13)(14)(15). Using a fluorescent probe covalently linked to the GT dinucleotide, this makes it possible to follow DNA binding and dinucleotide release, because both steps strongly influence molecular size of the fluorescent moiety.…”