Table 1.
Plasmids used in this work.
Fig 1.
Structural similarities between the N-terminal domains of AAV2-Rep and R388-TrwC.
(A) Topology diagram of the OBD domain of Rep (left) and relaxase domain of TrwC (right). The portion of the structure that forms the core active site typical of HUH endonucleases, consisting of five anti-parallel β-sheets and one α-helix and containing the active site tyrosine (helix E in AAV-Rep and helix A in R388-TrwC), is highlighted in red and blue, respectively. Helices with some degree of overlap are shown with their name (C and D in AAV-Rep, E and G in R388-TrwC). (B) Ribbon diagrams shown in the same orientation as in (A), with the active site tyrosine highlighted in yellow and the consensus HUH motif in green. TrwC has 2 active tyrosines, Tyr18 and Tyr26, but only Tyr18 is mapped in the TrwC structure. (C) Structural alignment of the HUH domains of Rep (red) and TrwC (blue) showing a high degree of similarity between the active sites of the two proteins, while no similarities are noted in the rest of the protein structure (shown in orange and violet, respectively).
Fig 2.
Design and validation of the TrwC/Rep chimeric protein.
(A) Schematic representation of Rep68, TrwC and the TrwC/Rep chimera. For each protein, overall configuration and functional domains are shown. The position of the HUH and Y2 motif, and the NLS (black bar) are indicated. Amino acid (aa) positions are indicated above each protein. (B) Western blot analysis of Rep68 and TrwC/Rep chimera expressed in 293T cells. Molecular weight in kDa is shown on the right. Antibodies used to detect proteins are shown on the left. (C) Localization of TrwC, Rep68 and TrwC/Rep chimera in 293T cells visualized by immunofluorescence microscopy. Images are shown at 60X magnification. (D) Assessment of the purity of His-Rep68 and His-TrwC/Rep chimera purified by nickel affinity and gel filtration chromatography. 200 ng of protein was loaded onto a 12% SDS-PAGE gel and subsequently stained with Coomassie Brilliant blue to visualize the quality of the purified protein. Marker (M) is shown on the left; molecular weight from top to bottom is: 250, 150, 100, 75, 50 kDa.
Fig 3.
DNA helicase activity of Rep68 and TrwC/Rep.
Increasing amounts of Rep68 and TrwC/Rep chimeric protein (0, 1, 10, 100 ng of protein) were assayed in the presence or absence of ATP. “boil” indicates heat-denatured substrate, used as a positive control. Products of the reaction were resolved on 12% native polyacrylamide gels. The diagram to the left of the gel shows the position of the substrate (partial duplex M13 DNA) and unwound labelled ssDNA. The percent of unwound substrate was quantified using the Image Quant TL software. The helicase efficiency percentage is a representative example obtained from 3 independent experiments (n = 3).
Fig 4.
DNA binding activity of Rep68 and TrwC/Rep.
(A) Rep68 and TrwC/Rep DNA substrates used for binding assays. Rep68 substrates were an AAV ori heteroduplex, and an equivalent AAV ori substrate mutated in the RBS sequence (RBSmut). trs is shown in boldface; nicking site is indicated by a slash. RBS is highlighted in bold italic. The AAVS1 minimal sequence (30) is presented below. TrwC/Rep chimera substrates were oligonucleotides oriTw(25+8) and oriTw(25+8) mutated in the inverted repeat IR (IRmut). Horizontal lines and nucleotides highlighted in boldface show sequence requirements for binding and nicking activities [77]. The nic site is represented by a slash. The IR recognized during binding is indicated with arrows; distal and proximal arms are shown. * represents the radioactively labelled strand. (B) and (C) EMSA assays with His-Rep68 and His-TrwC/Rep chimera, respectively. 30 fmol of the indicated radiolabelled substrates were incubated either with 100 ng of His-Rep68 or 200 ng of His-TrwC/Rep. All reactions contain 400 ng of poly(dI-dC) as nonspecific DNA. Competition assays were done using cold competitor DNA at 10- to 90-fold molar excess. Products were analysed on a native 6% polyacrylamide gel. Percentage of bound substrate (“% shift”) was calculated using Image Quant TL software. Bound and unbound products of the reaction are indicated with arrows. The DNA binding percentage is a representative example obtained from 3 independent experiments (n = 3). (D) Fluorescence polarization assay with oriTw(25+8) DNA labelled with carboxyfluorescein at 5 mM concentration. Binding was performed in 25 mM HEPES (pH 7.0), 200 mM NaCl at room temperature. Data analysis was performed as described by Yoon-Robarts et al. [57].
Fig 5.
Rep68 and TrwC/Rep nick sc plasmid DNA containing binding and nicking sites.
(A) Schematic representation of the scDNA nicking assay. When incubating a sc plasmid with an endonuclease, the protein nicks specifically inducing relaxation of the plasmid (open circular, oc), which will result in slower migrating DNA species when visualised on an agarose gel. (B) His-Rep68 and His-TrwC/Rep-mediated scDNA nicking. 100 ng of the plasmid containing the target sequence indicated on top of the gels was incubated as described in Materials and Methods with either purified His-Rep68 (left panels) or His-TrwC/Rep chimera (right panels). Products were resolved on 1% agarose gels, subsequently stained with ethidium bromide. “-“: sc plasmid incubated in the reaction buffer and processed as described above, but in the absence of protein. sc and oc products are indicated with arrows. The oc/sc ratios of quantified DNA bands are indicated at the bottom of the panels. The presented gels are representative of 3 independent experiments (n = 3).
Fig 6.
Sedimentation velocity analysis of the TrwC/Rep chimera.
(A) Sedimentation profiles of the TrwC/Rep chimera at three different concentrations of protein– 2.5, 5 and 10 μM—in 200 mM NaCl (top) or 500 mM NaCl (bottom), obtained using the SEDFIT programme. (B) Sedimentation profile of TrwC/Rep incubated with its specific substrate oriTw(25+8) oligonucleotide. Top: in the absence of DNA, the substrate sediments with a sedimentation coefficient of 3.8S; Bottom: at a 4:1 protein:DNA ratio, the analysis shows a peak of 12.7S, suggestive of hexamer species based on the theoretical S20,w values obtained as described before [36].
Fig 7.
Site-specific integration assay.
(A) Scheme of the plasmids used in the assay and the cointegrate molecule obtained. The donor suicide plasmid pCMS11 (pR6K::oriTp oriTw) is represented with a dotted line (left panel). oriTp and oriTw, the origins of transfer recognized by the relaxases of plasmids RP4 and R388 respectively, are shown as boxes in grey and black, respectively. nic sites are indicated with triangles in the respective colour code. The chloramphenicol resistance gene is represented as a white arrow (CmR). Recipient plasmid (pLA32) containing oriTw is shown as a black line (center panel). The last plasmid is the result of the integration reaction and is referred to as the cointegrate molecule (right panel). Relevant restriction sites are indicated in each plasmid. P1 and P2 are the oligonucleotides used to amplify the DNA of the integrants. (B) Representation of the integration assay in which the integrase is expressed in the recipient bacteria. The suicide plasmid is mobilized by the TraI relaxase (grey diamond) from the RP4 conjugative system (represented by a square) in a S17.1λpir donor strain to a DH5α recipient harbouring the oriTw-containing plasmid. Four different assays are represented: (i) no expression of integrases in the recipient bacteria (negative control); (ii-iv) recipient bacteria contain a plasmid coding for the relaxase domain of TrwC (N293-TrwC), full-length TrwC, or the TrwC/Rep chimera, respectively. TrwC protein is represented as black ellipses, and the Rep domain, in light gray. Integration frequencies obtained are also shown in the figure. Data are the mean of five independent experiments. Frequency is given as integrants/donors. (C) PCR analysis using primers P1 and P2. Agarose gel showing PCR amplicons (1.2 kb) obtained as a result of the integration reaction mediated by TrwC or TrwC/Rep chimera. RP, recipient plasmid; DP, donor plasmid; HP1, helper plasmid coding for TrwC protein; HP2, helper plasmid for TrwC/Rep chimera expression; M (1kb ladder): 10-8-6-5-4-3-2-1.5–1 kb.
Table 2.
Site-specific integration mediated by Rep, TrwC and TrwC-Rep using and episomal assay.