C-terminal phenylalanine of bacteriophage T7 single-stranded DNA-binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA

Abstract

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.

FIGURE 1.

Amino acid changes in gp5 suppressor mutant polymerase(s). The amino acid changes in gp5 arising from the suppressor mutations in gene 5 are identified in the crystal structure of gp5/trx in complex with a primer-template and a nucleoside triphosphate (31). gp5 (light gray), trx (dark gray), and primer/template (red) are depicted. The suppressor mutation G371K (gp5-sup1) is shown in yellow and T258M and A411T (gp5-sup2) in orange.

FIGURE 2.

DNA synthesis and exonuclease activity catalyzed by wild-type gp5/trx, gp5-sup1/trx, and gp5-sup2/trx. A, rates of DNA synthesis. A 24-nt oligo annealed to circular M13 ssDNA (20 nm) was used as the primer-template in a standard polymerase assay with gp5/trx (■), gp5-sup1/trx (△), or gp5-sup2/trx (□) (0.3 nm). The rate of incorporation (incorp.) of [³H]dTMP was measured at 37 °C on DE81 filter disks as described under “Experimental Procedures.” The data have been presented as the rates of incorporation of total deoxyribonucleoside monophosphate. The length of the template is 9950 bases. B, 3′–5′-exonuclease activity on M13 dsDNA. Uniformly ³H-labeled M13 dsDNA was prepared by annealing a 24-nt oligonucleotide to M13 DNA and then extending the primer using gp5/trx in the presence of [³H]dTTP. Acid-soluble radioactivity was measured as described under “Experimental Procedures.” The percentage of DNA hydrolyzed has been presented as a function of polymerase concentration (conc.). 100% dsDNA corresponds to 0.5 nmol of M13 DNA (in terms of total nucleotides). wt, wild type.

FIGURE 3.

Binding of gp5-sup1/trx to gp2.5. The interaction of gp5 with gp2.5 as measured by surface plasmon resonance. gp2.5 (150 response units (RU)) was immobilized on a Biacore carboxymethyl-5 chip, and increasing concentrations (conc.) of gp5-sup1/trx were flowed over the surface of the chip. A, sensorgrams of the binding of gp5-sup1/trx (0.002–16 μm) to gp2.5. Only 9 of 14 concentrations tested are shown for clarity. B, K_D determination of the binding of gp5-sup1/trx to gp2.5. Data points represent the equilibrium average response for the last 10 s of the injection in each of the experiments shown in A, where steady-state conditions have been obtained. The K_D of 1.1 μm was calculated using the steady-state fit model provided by BIAEVAL 3.0.2 software (Biacore). It should be noted that the calculated K_D value is apparent and not absolute due to the random immobilization of gp2.5 on the surface of the chip with some molecules having a conformation that may not support interaction with the polymerase.

FIGURE 4.

Effect of gp2.5 on strand displacement DNA synthesis catalyzed by gp5/trx at a nick in DNA. Strand displacement synthesis catalyzed by gp5/trx on nicked DNA was monitored by coupling the reaction to leading strand synthesis mediated by gp5/trx and DNA helicase. A, strand displacement synthesis on M13 nicked circular DNA and M13 circular dsDNA bearing a 5′-ssDNA tail of 36 nucleotides (see inset). Each reaction contained 10 nm DNA, 10 nm helicase, and the indicated amounts of gp5/trx (0–20 nm) in a total reaction volume of 10 μl. After incubation for 10 min at 37 °C, the amount of [³²P]dTMP incorporated (incorp.) into DNA was measured. B, effect of gp2.5 on strand displacement synthesis at a nick. The reaction with nicked DNA described in A was carried out with the addition of 4 μm gp2.5 or gp2.5-FD as indicated. C, effect of gp2.5 and gp2.5-FD on strand displacement synthesis catalyzed by gp5-sup2/trx. gp5-sup2/trx was substituted for wild-type gp5/trx in the reaction described in B, and the effect of wild-type gp2.5 or gp2.5-FD was examined. D, effect of gp2.5 and gp2.5-FD on strand displacement synthesis catalyzed by gp5-sup1/trx. gp5-sup1/trx was substituted for wild-type gp5/trx in the reaction described in B, and the effect of wild-type gp2.5 or gp2.5-FD was examined. E, radioactive products of strand displacement synthesis. The DNA products of the reaction shown in B and C for a single concentration (conc.) of gp5/trx (10 nm) were denatured and analyzed by electrophoresis on 0.6% alkaline-agarose gel.

FIGURE 5.

Effect of gp2.5 on coordinated DNA synthesis using mini-circle DNA. Coordinated synthesis of gp5/trx variants is studied in the presence of gp2.5 and helicase on a mini-circle DNA. The effect of gp2.5-FD on coordinated DNA synthesis was also examined on gp5/trx, gp5-sup1/trx, and gp5-sup2/trx. The leading and lagging strand synthesis of each polymerase is studied by formation of DNA products. The product of leading strand synthesis is more than 30,000 nucleotides that lie beyond the resolution of the gel. The product of lagging strand synthesis, Okazaki fragments, forms within a size range of 800–3000 nucleotides, beyond which the system loses the coordination with the leading strand polymerase. Marker, M, on left measures length of the Okazaki fragments. wt, wild type.

FIGURE 6.

Amino acid changes in gp5-sup1. The location of the amino acid change in gp5-sup1 arising from the suppressor mutations in gene 5 is identified in the crystal structure of the gp5/trx in complex with a primer-template and a nucleoside triphosphate (31). gp5 with trx is depicted in gray. The primer-template is shown in red. Hydrophobic residues are displayed in green, and aromatic residues are displayed in dark green (Trp-351 and Tyr-356). Positively charged residues are displayed in blue (Glu-535 and Glu-546). Residues in direct contact with DNA (Val-364 and Asp-366) are circled in a black oval.

FIGURE 7.

Model for the role of gp2.5 in the initiation of leading strand synthesis at nicks in DNA. A, gp5/trx binds to 3′-hydroxyl at a nick in duplex DNA. Alternate hydrolysis of the DNA by the 3′–5′-exonuclease activity of gp5 and nucleotide polymerization allows gp5/trx to idle at the nick. B, transient breathing of the 5′ terminus at the nick allows ssDNA to give rise to 5′-ssDNA tail. C, gp2.5 binds to ssDNA tail, and the C-terminal tail of gp2.5 binds to gp5/trx to stabilize the complex and allow for limited strand displacement synthesis. D, when the 5′-tail is sufficiently long gp4 assembles as a hexamer and displaces gp2.5. gp4 and gp5/trx form a stable complex and initiate leading strand DNA synthesis.