Protein p56 from the Bacillus subtilis phage phi29 inhibits DNA-binding ability of uracil-DNA glycosylase

Abstract

Protein p56 (56 amino acids) from the Bacillus subtilis phage 29 inactivates the host uracil-DNA glycosylase (UDG), an enzyme involved in the base excision repair pathway. At present, p56 is the only known example of a UDG inhibitor encoded by a non-uracil containing viral DNA. Using analytical ultracentrifugation methods, we found that protein p56 formed dimers at physiological concentrations. In addition, circular dichroism spectroscopic analyses revealed that protein p56 had a high content of beta-strands (around 40%). To understand the mechanism underlying UDG inhibition by p56, we carried out in vitro experiments using the Escherichia coli UDG enzyme. The highly acidic protein p56 was able to compete with DNA for binding to UDG. Moreover, the interaction between p56 and UDG blocked DNA binding by UDG. We also demonstrated that Ugi, a protein that interacts with the DNA-binding domain of UDG, was able to replace protein p56 previously bound to the UDG enzyme. These results suggest that protein p56 could be a novel naturally occurring DNA mimicry.

Figure 1.

Analytical ultracentrifugation profile of protein p56. Sedimentation equilibrium profile of 75 μM p56 taken at 30 000 r.p.m., 20°C and at a wavelength of 280 nm. Grey circles represent the experimental data; the continuous line is the best fit M_w (13 000 ± 360); the discontinuous line is the theoretical gradient of a p56 monomer. The residuals to the fit are shown in the upper part of the figure. Insert: Sedimentation velocity (60 000 r.p.m., 20°C) distributions of the same p56 preparation showed in the main figure.

Figure 2.

CD spectrum of native p56 in the far-UV region at 20°C. Protein p56 (0.7 mg/ml) was in 20 mM NaH₂PO₄, pH 8.0. Experimental data were acquired using 0.1-mm optical path length quartz cells.

Figure 3.

Temperature-associated changes in the secondary structure of p56. (A) Temperature CD transition curve of p56 in 20 mM HEPES, pH 8.0, measured at 218 nm, between 15 and 60°C. The continuous line represents the best fit of a two state model to the experimental data. (B) CD spectra of p56 in the far-UV region at the indicated temperatures. 1-mm optical path length quartz cells were used.

Figure 4.

Protein p56 inhibits E. coli UDG activity. The 5′-end ³²P-labelled ssDNA-U¹⁶ substrate (S) (1.3 nM) was incubated with UDG (5 nM) in the absence or presence of p56. After 8 min, the reaction mixtures were treated with NaOH. Formation of the cleavage product (P) was monitored by autoradiography after resolution on 8 M urea/20% polyacrylamide gels.

Figure 5.

Effect of urea on the stability of preformed UDG–p56 complexes. UDG (1.9 μM) was incubated with either p56 (6.4 μM) or Ugi (4.2 μM; New England Biolabs) at room temperature for 15 min, and kept at 4°C for 15 min to allow formation of UDG–p56 and UDG–Ugi complexes. Then, the reactions were incubated with the indicated amount of urea for 30 min at room temperature, and analysed by basic-native PAGE (16% polyacrylamide). Gel electrophoresis was performed at 4°C. The gel was stained with SyproRuby (Molecular Probes).

Figure 6.

Protein p56 prevents UDG from binding to DNA. (A) UDG was applied to a ssDNA affinity column. The enzyme was neither detected in the flow-through (FT) nor in the washing steps (W). Protein p56 was used to elute UDG bound to the column. The elution fractions (E1 to E7) were separated by SDS-Tricine-PAGE. (B) Preformed UDG–p56 complexes were loaded onto a DNA affinity column. Purified UDG (1.8 μg) and protein p56 (2 μg) were run in the same gel (lane C). The amount of both UDG (white circle) and p56 (black circle) in each fraction was determined by densitometric scanning of the gel stained with Coomassie Blue.

Figure 7.

Protein p56 inhibits DNA-binding ability of UDG. Electrophoretic mobility shift assays were performed in the absence or in the presence of the indicated proteins. A radiolabelled dsDNA fragment (121 bp) containing uracil in place of thymine residues was used as substrate.

Figure 8.

Alignment of the p56 region spanning amino acids Asp11 and Leu56 with the Ugi region spanning residues Asn3 and Glu49. Grey boxes enclose amino acids that are identical or conserved in both proteins. The X-ray crystal structure of Ugi in complex with E. coli UDG has been determined (18). According to this structure, the UDG Leu191 side-chain makes contacts with eight hydrophobic side-chains of Ugi (Met24, Val29, Val32, Ile33, Val43, Met56, Leu58 and Val71). Closed circles indicate Ugi residues that belong to such hydrophobic-binding pocket. Open circles denote some of the hydrogen-bonding interactions between Ugi and E. coli UDG (Gln19, Glu20, Ser21, Leu23, Glu27, Glu28, Thr45, Asp61, Asp62, Tyr65 and Gln73).

Figure 9.

Ugi replaces protein p56 previously bound to UDG. UDG (1.9 μM) and protein p56 (6.4 μM) were incubated at room temperature for 15 min, and kept at 4°C for 15 min (formation of UDG–p56 complexes). Then, Ugi was added (4.2 μM) or not to the reaction mixture. After 10 min at room temperature, samples were analysed by basic-native PAGE (16% polyacrylamide). Gel electrophoresis was performed at 4°C. The gel was stained with SyproRuby.