Characterization of bacteriophage T4-coordinated leading- and lagging-strand synthesis on a minicircle substrate

Abstract

The DNA replication complex of bacteriophage T4 has been assembled as a single unit on a minicircle substrate with a replication fork that permits an independent measurement of the amount of DNA synthesis on both the leading and lagging strands. The assembled replisome consists of the T4 polymerase [gene product 43 (gp43)], clamp protein (gp45), clamp loader (gp44͞62), helicase (gp41), helicase accessory factor (gp59), primase (gp61), and singlestranded DNA binding protein (gp32). We demonstrate that on the minicircle the synthesis of the leading and lagging strands are coordinated and that the C-terminal domain of the gp32 protein regulates this coordination. We show that the reconstituted replisome encompasses two coupled holoenzyme complexes and present evidence that this coupling might include a gp43 homodimer interaction. Bacteriophage T4 is one of the more elementary replication systems using eight proteins in the formation and propagation of a replication fork. Central to the replication process, the T4 DNA polymerase [gene product 43 (gp43)] catalyzes nucleotide incorporation in a 5Ј to 3Ј direction whereas its 3Ј to 5Ј exonuclease activity serves to maintain fidelity during replication (1). Alone, the T4 polymerase will incorporate nucleotides in a distributive fashion, and, to avert quick dissociation from the DNA, interactions between the gp43 polymerase and accessory factors are essential (2, 3). These accessory factors include the gp45 protein (sliding clamp), which possesses a ring-shaped structure with an internal diameter large enough to encircle DNA, and the 44͞62 protein complex, which loads the sliding clamp onto DNA (4, 5). In addition, a DNA helicase (gene product 41) is required for the ATP-or GTP-dependent unwinding of the duplex DNA to be replicated at the front of the replication fork (6). The T4 single-stranded DNA binding protein gp32 is needed to prevent the reannealing of duplex DNA at the replication fork (7). The helicase has its own accessory factor, the gene product 59 protein, which is required to load the gp41 helicase onto single-stranded DNA coated with the gp32 single-stranded DNA binding protein (8). Finally, associated with the helicase is the DNA primase (gp61), which provides the pentaribonucleotide primers needed for the initiation of laggingstrand DNA synthesis (9).The dynamics of holoenzyme (gp43, gp45, and gp44͞62) assembly have been addressed both kinetically and structurally (10). The holoenzyme assembly process proceeds in a ordered fashion in which the gp45 sliding clamp is first loaded onto the DNA by the 44͞62 protein complex in an ATP-dependent process followed by the rapid association of the polymerase (gp43), with the 45 clamp mediated through the carboxyl terminus of the polymerase (11, 12). Once formed, the holoenzyme is stable with a dissociation rate constant (k off ) of 0.01 sec Ϫ1. The same basic replication strategy is shared by all DNA polymerases characterized to date in which DNA synthesis occurs by the successive addition of nucleotides to the 3Ј ...

Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

“…Interestingly, T4 DNA polymerase forms dimers. The fingers of T4 DNA polymerase are also involved in mediating the protein-protein interaction (38), suggesting an analogous mechanism for coordination of DNA synthesis in the phage T4 replication system.…”

Honey, I shrunk the DNA: DNA length as a probe for nucleic‐acid enzyme activity

“…Different models have been put forward to explain how these slow enzymatic steps can take place at the lagging strand without losing coordination with the continuous and rapid leading-strand synthesis. [80][81][82] Lee et al used single-molecule techniques to directly interrogate the kinetics of leading-and lagging-strand synthesis. 35 The authors used the replication machinery of the T7 bacteriophage, a system that can be reconstituted in vitro with a small number of purified proteins (see Figure 4).…”

Conditional Coupling of Leading-strand and Lagging-strand DNA Synthesis at Bacteriophage T4 Replication Forks

“…In support of the model, they presented data showing that decreasing the polymerase concentration over a range of 34 -0.4 nM did not increase the size of Okazaki fragments, as would have been expected if DNA synthesis were uncoupled. Recently, further support for this model was obtained using a synthetic 70-nucleotide circle as a template for DNA synthesis catalyzed by T4 proteins (13). Coordinated synthesis of leading and lagging strands was observed with 200 nM exonuclease-deficient (D219A) gp43.…”

“…Such complexes would also increase the difference in incorporation between the diluted and undiluted reactions. A recent report (13) analyzed DNA replication catalyzed by D219A exonuclease-deficient gp43 plus the other seven T4 proteins using a 70-nucleotide minicircle substrate. This minicircle lacks dG residues.…”

“…A, Sequenase at 47.5 nM was incubated alone (lanes 1-3) or in the presence of the indicated T4 proteins (lanes 4 -15) for the indicated times in the standard replication mixture. CTP and UTP were omitted from the reactions shown in lanes [13][14][15]. B, total dGMP incorporation in reactions with T4 DNA polymerase holoenzyme or Sequenase.…”