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A257T Linker Region Mutant of T7 Helicase-Primase Protein Is Defective in DNA Loading and Rescued by T7 DNA Polymerase

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A257T Linker Region Mutant of T7 Helicase-Primase Protein Is Defective in DNA Loading and Rescued by T7 DNA Polymerase

Gayatri Patel et al. J Biol Chem.

Abstract

The helicase and primase activities of the hexameric ring-shaped T7 gp4 protein reside in two separate domains connected by a linker region. This linker region is part of the subunit interface between monomers, and point mutations in this region have deleterious effects on the helicase functions. One such linker region mutant, A257T, is analogous to the A359T mutant of the homologous human mitochondrial DNA helicase Twinkle, which is linked to diseases such as progressive external opthalmoplegia. Electron microscopy studies show that A257T gp4 is normal in forming rings with dTTP, but the rings do not assemble efficiently on the DNA. Therefore, A257T, unlike the WT gp4, does not preassemble on the unwinding DNA substrate with dTTP without Mg(II), and its DNA unwinding activity in ensemble assays is slow and limited by the DNA loading rate. Single molecule assays measured a 45 times slower rate of A257T loading on DNA compared with WT gp4. Interestingly, once loaded, A257T has almost WT-like translocation and DNA unwinding activities. Strikingly, A257T preassembles stably on the DNA in the presence of T7 DNA polymerase, which restores the ensemble unwinding activity of A257T to ∼75% of WT, and the rescue does not require DNA synthesis. The DNA loading rate of A257T, however, remains slow even in the presence of the polymerase, which explains why A257T does not support T7 phage growth. Similar types of defects in the related human mitochondrial DNA helicase may be responsible for inefficient DNA replication leading to the disease states.

Figures

FIGURE 1.
FIGURE 1.
Structure of T7 gp4 and location of the A257T mutation. A, T7 gp4 hexameric ring showing the arrangement of the helicase and primase domains (Protein Data Base ID code 1Q57). B, One subunit of the gp4 ring showing the 26-amino acid linker region between the C-terminal helicase and N-terminal primase domains. Ala257 is present in the linker region. C, close-up view of the Ala257 residue in relationship to proximal Gln253 and Val230. D, close-up view of A257T mutation showing clashes of Thr257 with the backbones of residues Gln253 and Val230.
FIGURE 2.
FIGURE 2.
Helicase activities. A, scheme showing the ensemble unwinding assay. The ring-shaped hexameric helicase loads on the 5′-overhang and moves from the 5′-end to the 3′-end to unwind the fluorescein-labeled dsDNA fork substrate. T7 gp4 WT or A257T (100 nm) was preincubated with the fluorescent ds30 (23%) substrate (10 nm) and dTTP (2 mm) in one syringe of the stopped-flow instrument, and the unwinding reaction was initiated by addition of MgCl2 from a second syringe at 25 °C. B, unwinding kinetics of WT gp4. The lag kinetics was fit to the incomplete gamma function (29) to obtain a rate of ∼67 bp/s for the WT gp4. C, fluorescence signal resulting from unwinding of dsDNA into ssDNA by A257T plotted against time. The kinetics fit to a single exponential rate constant of 1.3 × 10−3 s−1. D, scheme of the single molecule optical trap setup (not to scale). A helicase/primase molecule separates the dsDNA, while a constant force is maintained on the DNA fork junction by adjusting the position of the glass coverslip relative to the optically trapped microsphere. The displacement of the trap relative the coverslip is utilized to determine the amount of DNA unwound (and subsequently the unwinding rate). E, comparison of ssDNA translocation rate of WT gp4 and A257T. Experiments were carried out at 0.3 nm WT and 30 nm A257T, 2 mm dTTP, 5 mm MgCl2. F, comparison of the unwinding rate WT gp4 and A257T. G, comparison of the initiation times of DNA unwinding by WT gp4 and A257T. The initiation times were experimentally determined at 5 nm hexamer, 2 mm dTTP, 5 mm MgCl2. The data represent average from >20 traces.
FIGURE 3.
FIGURE 3.
Electron microscopy studies of WT and A257T gp4. A, WT T7 gp4A or A257T 2.1 μm was incubated at 24 °C in triethanolamine buffer, pH 7.5, with 1.3 mm dTMPPCP, 2 mm magnesium acetate, 0.6 nm M13 ssDNA. B, WT or A257T was incubated at 24 °C with 2 mm dTTP, 7 mm MgCl2, 3 mm EDTA, 0.6 nm M13 ssDNA. C, WT or A257T was incubated with 2 mm dTTP and 3 mm EDTA. D, WT or A257T was incubated with 2 mm dTTP, 3 mm EDTA, and 0.6 nm M13 ssDNA.
FIGURE 4.
FIGURE 4.
DNA unwinding by mixed A257T-WT hexamers. A, WT gp4 (20 nm) was preincubated with various concentrations of A257T (10–100 nm) to make mixed hexamers, which were loaded on the fluorescein-labeled ds30 (23%) forked unwinding substrate with dTTP. The unwinding kinetics was measured by the stopped-flow fluorescence unwinding assay. B, the unwinding time traces show the decrease in amplitude with increasing fraction of A257T in the mixed hexamers. The kinetics was fit to the incomplete gamma function (29) to obtain the unwinding rates and amplitudes. C, the unwinding rate (filled circles) and amplitude (open circles) are plotted against a fraction of A257T.
FIGURE 5.
FIGURE 5.
Rolling circle DNA synthesis by WT and A257T gp4 in the presence of T7 DNA polymerase. A, schematic shows the 70-bp minicircle replication fork. B, WT or A257T gp4 at 100 nm was incubated with 100 nm T7 DNA polymerase (T7 gp5-thioredoxin) and minicircle DNA (50 nm), and DNA synthesis was measured using [α-32P]dGTP in the presence of all dNTPs. The DNA products are resolved by electrophoresis on a 1% alkaline-agarose gel. Images show a time course of increasing kilobase pair length of DNA synthesis by WT and A257T in presence of T7 DNA polymerase.
FIGURE 6.
FIGURE 6.
T7 DNA polymerase rescues the DNA unwinding defect of A257T gp4. A, T7 DNAP and WT or A257T gp4 were assembled on the radiolabeled ds40 (20%) replication fork with dTTP. Unwinding reaction was initiated with MgCl2. DNA unwinding proceeds either by both proteins moving together, forming a loop-like structure, or helicase moving alone. B, native polyacrylamide gels show the unwinding kinetics of WT and A257T gp4 in the presence and absence of T7 DNAP. C, unwinding kinetics by WT and A257T in the presence (open circles) and in the absence of T7 DNAP (filled circles) is shown. WT gp4 unwinds the forked substrate in the absence of T7 DNAP at ∼82 bp/s in the presence of T7 DNAP at ∼65 bp/s. The unwinding rate of A257T gp4 in the presence of T7 DNAP is ∼50 bp/s.
FIGURE 7.
FIGURE 7.
Slow binding of A257T to the replication fork. A, a mixture of WT or A257T gp4 (200 nm), T7 DNAP (200 nm), dTTP (4 mm), and radiolabeled ds40 (20%) replication fork substrate was incubated for various time intervals (Δt: 5 s to 30 min). A solution of MgCl2 and dT90 trap was then added to initiate the unwinding reaction, which was quenched in 5 s. The DNA substrate and the unwound ssDNA were resolved by native-PAGE. B, the image shows the extent of unwinding in 5 s in reactions containing T7 DNAP alone or T7 DNAP plus A257T or WT gp4 as a function of Δt. C, fraction of replication fork unwound as a function of Δt. The data were fit to single exponential equation to obtain the rate of helicase assembly in the presence of T7 DNAP: WT (filled circles) at 0.03 s−1 and A257T (open circles) at 0.001 s−1.

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