A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

Abstract

The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 singlestranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.DNA synthesis is initiated on the lagging strand of the replication fork by a site-specific RNA polymerase known as a DNA primase (1). The primase synthesizes a short RNA primer that is extended by DNA polymerase to create an Okazaki fragment (2, 3). During replication, interactions between the primase and other replicative proteins such as DNA polymerase, DNA helicase, and single-stranded DNA (ssDNA) 1 -binding protein are required to initiate DNA synthesis. It has been difficult to identify these essential interactions because they occur transiently and involve weak interactions between multiple partners.We have been studying the physical and functional interactions between proteins of the comparatively simple bacteriophage T7 replication system. Phage T7 replicates DNA using four proteins, T7 gene 4 primase-helicase protein (gp4), T7 gene 2.5 ssDNA-binding protein (gp2.5), and T7 DNA polymerase (gp5) complexed with its processivity factor, Escherichia coli thioredoxin (4). The reactions catalyzed by the individual proteins are coordinated during replication by the assembly of a multiprotein complex that moves with the replication fork (5, 6). During replication, the primase-helicase directly contacts both the DNA polymerase and gp2.5 (7-10). A C-terminal acidic segment of the primase-helicase is required for its interaction with the DNA polymerase (10). An interaction between gp2.5 and T7 DNA polymerase is required for the coordinated synthesis of both leading and lagging strands of the replication fork (5, 6). A C-terminal 21-residue region of gp2.5 is essential not only for the interactions with the DNA polymerase and the primase-helicase but also for dimerization of gp2.5 (9). Although many pairwise interactions between T7 replication proteins have been identified, the overall physical arrangement of subunits and their stoichiometries in the replication complex are unknown.Crystal structures of all the individual T7 replication proteins (11-16) have been determined, and their enzymatic ac...

Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome

“…The enhanced use of primers by full-length gp4 suggests that the polymerase engages gp4 through contacts with the primase and helicase domains or the helicase domain efficiently tethers the primase domain to DNA. The requirement for a physical interaction between gp4 and T7 DNA polymerase to promote primer extension (14,15) is underscored by the inability of other polymerases, such as T4 DNA polymerase or the Klenow fragment of E. coli DNA polymerase I, to extend primers synthesized by gp4 (Fig. 1B).…”

An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

“…In comparison, gp4⌬primase binds the ssDNA 3-fold less tightly (K d of 411 Ϯ 53 nM) and binds 30% of the oligonucleotide at the highest protein concentration. The 3-fold poorer binding of gp4⌬primase to ssDNA is likely due to the fact that gp4⌬primase does not contain the RPD that alone can bind ssDNA (31). It was proposed earlier that the primase facilitates loading of gp4 onto ssDNA (32).…”

The Roles of Tryptophans in Primer Synthesis by the DNA Primase of Bacteriophage T7

“…In a previous study, the replacement of Trp-42 with alanine in the ZBD decreased de novo synthesis of tetraribonucleotide and partially reduced its utilization as primers by DNA polymerase (24). However, replacement of tryptophan with alanine is too drastic to meaningfully deduce the structural roles of the tryptophan from the properties of the altered ZBD.…”

“…1): Trp-42 in the ZBD, Trp-69 and Trp-97 in the N-terminal subdomain of the RPD, and Trp-147 and Trp-255 in the C-terminal TOPRIM fold of the RPD. A previous study showed that the replacement of Trp-42 with alanine in the ZBD greatly decreases tetraribonucleotide synthesis and partially reduces their utilization as primers by T7 DNA polymerase (24). In another study, the substitution of non-aromatic residues for Trp-69 significantly impairs the ability of gp4 to synthesize primers and deliver them to DNA polymerase (25).…”