Figure 4: The effect of macromolecular crowding on gp5/trx and gp4 interactions.

The three proteins move the replication fork at a rate of approximately 150 nt per second at 25°. The bifunctional gene 4 helicase-primase (gp4) assembles on the lagging strand as a hexamer, where it forms a complex with gp5/trx and unwinds the DNA duplex. (a) Effect of PEG on strand-displacement DNA synthesis mediated by gp5/trx and gene 4 helicase (gp4B). Using M13 dsDNA with a 5′-ssDNA tail (top), the efficiency of strand-displacement DNA synthesis in the presence of PEG was determined. The standard reaction contained the dsM13 template (10 nM), 0.3 mM dATP, dGTP, dCTP and [α-³²P] dTTP (0.1 μCi), 10 nM gp5/trx, 200 nM monomeric concentrations of gp4B and increasing amounts of PEG 1 kDa (0–8%). After incubation for 30 min at 37 °C and the amount of DNA synthesis was determined by the amount of [α-³²P] dTTP incorporated into DNA. (b) Primase-dependent DNA synthesis. The reaction was similar to a except that gp4A replaced gp4B and 10 nM M13 ssDNA replaced the dsM13 DNA. The reaction buffer also contained ATP and CTP (100 μM each). The amounts of primase-dependent DNA synthesis was determined by measuring the incorporation of [³²P] dTMP into DNA. The error bars were derived from three independent experiments.