Fig 1.
Structural prediction of full-length pUL89.
(A) The ribbon representation of full-length pUL89 model. (B) Surface charge representation of full-length pUL89 is shown. Region of positive, negative and neutral electrostatic potential are indicated in blue, red and white, respectively. Arrows indicate the N and C-terminus.
Fig 2.
Purification of pUL89 from transfected HEK 293T cells.
(A) Characterization of the monoclonal antibody against pUL89 (mAbUL89). Mock-infected (lane 1) and infected HELF (lane 2), HEK 293T (lane 3), HEK 293T transfected with pcDNA-89 (lane 4) and purified pUL89 (lane 5) were separated by 10% SDS–PAGE and transferred to nitrocellulose. The immunoblot was reacted with mAbUL89. GAPDH served as a loading control. (B) Silver staining of purification of pUL89. Lane 1, cytosol; lane 2, flow; lane 3–4, wash fractions (60 mM and 250 mM imidazole), lane 5–8 fraction with eluted pUL89 (500 mM imidazole). (C) Immunoblot of purification of pUL89. Lane 1, cytosol; lane 2, flow; lane 3–4, wash fractions (60 mM and 250 mM imidazole), lane 5–8 fraction with eluted pUL89 (500 mM imidazole). Probes were subjected to SDS-Page followed by silver staining or immunoblot analysis with anti-pUL89 (mAbUL89). Markers (kDa) are indicated on the left, the position of pUL89 on the right.
Fig 3.
Nuclease activity of wild-type and mutant pUL89 proteins.
(A) Purified pUL89 (0.5 μM) together with treatment with BDCRB (0–15μM) and (B) purified wild-type pUL89 (0,5 μM) and mutants of pUL89 (0,5 μM; pUL89-D463A; pUL89-E534A; pUL89-R544A; pUL89-H565A; pUL89-H571A; pUL89-D651A) were analysed by nuclease activity assay. (A) Lane 1, 600 ng pUC-aseq; lane 2, incubation with restriction endonuclease Hind III, lane 3 incubated with pUL89, lane 4, incubated with pUL89 treated with 1.0 μM BDCRB; lane 5, incubated with pUL89 treated with 2.5 μM BDCRB; lane 6, incubated with pUL89 treated with 5.0 μM BDCRB; lane 7, incubated with pUL89 treated with 7.5 μM BDCRB; lane 8, incubated with pUL89 treated with 10 μM BDCRB; lane 9, incubated with pUL89 treated with 12.5 μM BDCRB; lane 10, incubated with 0.5 μM pUL89 treated with 15 μM BDCRB. (B) Lane 1, 600 ng pUC-aseq; lane 2, incubation with restriction endonuclease Hind III, lane 3, incubated with wild-type pUL89, lane 4, incubated with pUL89-D463A; lane 5, incubated with pUL89-E534A; lane 6, incubated with pUL89-R544A; lane 7, incubated with pUL89-H565A; lane 8, incubated with pUL89-H571A; lane 9, incubated with pUL89-D651A. After incubation with plasmid DNA at 37°C, all probes were treated with proteinase K (final concentration 1 μg/μl) at 65°C. The arrows indicated three different plasmid DNA forms: circular covalently closed molecules (ccc), open circular molecules and linear forms. The quantifications were performed with the software Phoretix 1D (BioSytematica) and shown below the image.
Fig 4.
Identification of amino acids required for DNA binding using colorimetric DNA binding assays.
The assays were performed with purified pUL89 or indicated purified mutants (pUL89-D463A; pUL89-E534A; pUL89-R544A; pUL89-H565A; pUL89-H571A; pUL89-D651A) and biotinylated DNA probe. (A) Extinction at 620 nm. Lane 1, pUL89 wild-type (control) and values in the presence of 20 nmolar excess of competitor; lane 2, pUL89-D463A, +/- 20 nmolar excess of competitor; lane 3, pUL89-E534A, +/-20 nmolar excess of competitor; lane 4, pUL89-R544A, +/- 20 nmolar excess of competitor; lane 5, pUL89-H565A,+/-20 nmolar excess of competitor; lane 6, pUL89-H571A +/-20 nmolar excess of competitor; lane 7, pUL89-D651A, +/- 20 nmolar excess of competitor. (B) Percentage of reduction of DNA binding in comparison to the control. Lane 1, pUL89 wild-type (control), +/- competitor; lane 2, pUL89-D463A, +/- competitor; lane 3, pUL89-E534A, +/- competitor; lane 4, pUL89-R544A, +/- competitor; lane 5, pUL89-H565A, +/- competitor; lane 6, pUL89-H571, +/- competitor; lane 7, pUL89-D651A, +/- competitor. Values represent mean ± SD from three independent experiments. * p-value ≤ 0.05.
Fig 5.
Predicted structure of the DNA binding motif of full-length wild-type pUL89.
The left figure shows a ribbon representation of full-length pUL89 and double stranded DNA. Residues 580–600, expected to be responsible for interaction with pUL56, are colored in red. The figure on the right represents a magnified view of the local structure from the boxed region highlighting the predicted functional motifs and including high-resolution data for the C-terminal part of pUL89 (Nadal et al.; 25). Nadal et al. (2010) identified the nuclease domain comprised of D463, E534 and D651 and alluded to a Mg2+ dependent mechanism (Mg2+ is shown in green). In contrast to this, by using the full-length pUL89, this report confirms D463 (cyan) as an amino acid required for nuclease activity and argine 544 as critical for binding double stranded DNA (shown in green).
Fig 6.
Purification of recombinant baculovirus expressed rpUL89.
Gel permeation chromatography of rpUL89. The elution profile has 4 peaks. Coomassie-staining and immunoblot with monospecific mAbUL89 of fractions 13 and 14 are shown in the box. Molecular mass markers are indicated on the left, the position of pUL89 on the right.
Fig 7.
3-D reconstruction from single particle analysis of rpUL89.
Surface-rendered presentations of the rpUL89 3D reconstruction are shown face-on from the front (A,B), and side-on to the right (C,D) with (A, C) and without (B, D) the predicted structure of pUL89. The scale bar corresponds to 5 nm. The magenta colored segment represents the C-terminal domain and the blue colored the N-terminal domain of pUL89.