In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans

Abstract

Nucleotide excision repair (NER) is a universal and versatile DNA repair pathway, capable of removing a very wide range of lesions, including UV-induced pyrimidine dimers and bulky adducts. In bacteria, NER involves the sequential action of the UvrA, UvrB and UvrC proteins to release a short 12- or 13-nucleotide DNA fragment containing the damaged site. Although bacterial NER has been the focus of numerous studies over the past 40 years, a number of key questions remain unanswered regarding the mechanisms underlying DNA damage recognition by UvrA, the handoff to UvrB and the site-specific incision by UvrC. In the present study, we have successfully reconstituted in vitro a robust NER system using the UvrABC proteins from the radiation resistant bacterium, Deinococcus radiodurans. We have investigated the influence of various parameters, including temperature, salt, protein and ATP concentrations, protein purity and metal cations, on the dual incision by UvrABC, so as to find the optimal conditions for the efficient release of the short lesion-containing oligonucleotide. This newly developed assay relying on the use of an original, doubly-labelled DNA substrate has allowed us to probe the kinetics of repair on different DNA substrates and to determine the order and precise sites of incisions on the 5' and 3' sides of the lesion. This new assay thus constitutes a valuable tool to further decipher the NER pathway in bacteria.

Fig. 1. Deinococcus radiodurans UvrABC system.

a SDS-PAGE analysis of the purified drUvrA1, drUvrA2, drUvrB, and drUvrC proteins from D. radiodurans. The first lane corresponds to molecular weight markers in kDa. b Schematic diagram illustrating the design of the dsDNA substrates used in this study and the sites of incision by drUvrABC on the 5′ and 3′ sides of the lesion, corresponding to a fluorescein-conjugated thymine (green). An additional red fluorophore was added to the 5′ end of the substrates to allow to differentiate the DNA fragments released on the 5′ and 3′ sides of the lesion. c TBE-polyacrylamide urea-gel analysis of the drUvrABC incision activity in the presence or absence of each of the three Uvr proteins or ATP. Reactions were performed for 1 hour at 37 °C using 25 nM F26-seq1 substrate and different combinations of drUvrA1 (1 µM), drUvrB (0.5 µM), and drUvrC (2 µM) in the presence of 2.5 mM Mg²⁺ and 2.5 mM ATP. d TBE-polyacrylamide urea-gel analysis of the drUvrABC incision activity in reactions containing either 1 µM drUvrA1 or 1 µM drUvrA2. Reaction conditions were the same as in (c). The major bands observed by electrophoresis using either the green- or red filter are indicated with arrows. The large band indicated with a * in the red channel corresponds to the sample-loading dye that produces a strong fluorescence in the red filter. c–d Green-boxed gels were visualized with the green filter to detect fluorescein-labeled bands, whereas red-boxed gels were visualized with the red filter to detect ATTO633-labeled bands. Left lane: molecular weight marker composed of fluorescein-labeled oligonucleotides ranging from 10 to 50 bp. e Effect of drUvrA2 on the incision reaction performed by drUvrABC. Reactions were performed at 37 °C for 45 min using 25 nM F26-seq1 substrate, 0.25 µM drUvrA1, 0.5 µM drUvrB and 2 µM drUvrC (blue), supplemented with 0 (ratio 1:0), 0.25 (ratio 1:1), 0.5 (ratio 1:2), 1 (ratio 1:4), or 2 µM (ratio 1:8) drUvrA2. All reactions contained 2.5 mM MgCl₂ and were started by addition of 2.5 mM ATP. Dot-plots present the mean amount of 12 mer product (nM) released and standard deviation of four individual replicates illustrated as individual dots.

Fig. 2. Optimization of the in vitro NER system.

a Dual-incision activity by drUvrABC as a function of MgCl₂ concentration. Reactions were performed at 37 °C using 25 nM F26-seq1 substrate, 0.25 µM drUvrA1, 0.5 µM drUvrB and 2 µM drUvrC supplemented with 0, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, or 10.0 mM MgCl₂. Reactions were started by addition of 2.5 mM ATP. The graph presents the mean amount of 12 mer product (nM) released after 30 minutes (black symbols) and the standard deviation of three individual replicates shown as open red circles. b Dual-incision activity by drUvrABC as a function of ATP concentration. Reactions were performed at 37 °C using 25 nM F26-seq1 substrate, 1 µM drUvrA1, 0.5 µM drUvrB and 2 µM drUvrC, and 2.5 mM MgCl₂. Reactions were started by addition of 0, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 or 10.0 mM ATP. The graph presents the mean amount of 12 mer product (nM) released after 45 minutes (black symbols) and the standard deviation of three individual replicates shown as open red circles. c Effects of metals on the incision activity by drUvrABC. Left: Dual incision activity in the presence of 2.5 mM of Mg (blue), Mn (red), Fe (green), Zn (purple), Cu (orange), Ni (black), or Co (brown). Reactions were performed at 37 °C using 25 nM F26-seq1 substrate, 1 µM drUvrA1, 0.5 µM drUvrB and 2 µM drUvrC. Reactions were started by addition of 2.5 mM ATP. The dot plots present the mean amount of 12 mer product (nM) released after 30 minutes and standard deviation of three individual replicates shown as filled circles. Right: Dual incision activity in the presence of 2.5 mM Mg alone (blue), or 2.5 mM Mg supplemented with 0.25 mM of Mn (red), Fe (green) Zn (purple), Cu (orange), Ni (black), or Co (brown). Reactions were performed at 37 °C using 25 nM F26-seq1 substrate, 1 µM drUvrA1, 0.5 µM drUvrB and 2 µM drUvrC. Reactions were started by addition of 2.5 mM ATP. The dot plots present the mean amount of 12 mer product (nM) released after 20 minutes and standard deviation of three individual replicates shown as filled circles. The dashed line indicates the extent of incision in the presence of Mg alone. d Effects of temperature on the drUvrABC incision activity. Time-course experiments were performed at 25 (green), 30 (purple), 37 (blue), and 42 °C (red) for 1 hour. The graph presents the mean amount of 12 mer product (nM) released at each timepoint (filled triangles) and standard deviation of at least three individual replicates shown as open circles. e Effects of salt on the drUvrABC incision activity. Dual incision activity by drUvrABC as a function of NaCl (blue) and KCl (red) concentration. Reactions were performed at 37 °C using 25 nM F26-seq1 substrate, 1 µM drUvrA1, 0.5 µM drUvrB and 2 µM drUvrC in reaction buffer containing 0, 10, 25, 50, 100, 200, or 300 mM NaCl or KCl. Reactions were started by addition of 2.5 mM ATP. The graph presents the amount of 12 mer product (nM) released after 45 minutes of reaction.

Fig. 3. MALDI-ToF mass spectra of the drUvrABC incision-reaction substrates and products.

The incision reactions were performed at 37 °C for 1 hour using 25 nM F26-seq1 substrate, 1 µM drUvrA1, 0.5 µM drUvrB and 2 µM drUvrC, 2.5 mM MgCl₂, and 2.5 mM ATP. Peaks corresponding to either substrates (a) or products (b) of the incision reaction are indicated with red arrows. Masses of major peaks are indicated in Da. In (a), the two peaks with m/z values close to 8000 correspond to the doubly charged forms of the starting oligonucleotides bearing the lesion (5′-ATTO633-F26-seq1) and its complementary strand (Rev-seq1).

Fig. 4. Kinetics of repair by drUvrABC.

a Time-course experiments following the changes in abundance of the different DNA fragments (50 mer in blue, 32 mer in green, 18 mer in red, and 12 mer in purple, illustrated to the right of the graph) as a function of time. The graph presents the mean (filled triangles) and standard deviation of six individual replicates shown as open circles. Reactions were performed at 37 °C using 25 nM F26-seq1 substrate, 1 µM drUvrA1, 0.5 µM drUvrB, and 2 µM drUvrC in the presence of 2.5 mM MgCl₂. Reactions were started with 2.5 mM ATP. The observed rate of product release, K_obs, corresponding to the rate of the dual-incision reaction, was determined to be 0.031 min⁻¹ by fitting the 12 mer data points to a single exponential model. The observed rate of 18 mer release, K_obs1, corresponding to the rate of the 5′ incision reaction, was determined to be 0.030 min⁻¹ by fitting the 18 mer data points to a single exponential model. These findings suggest that the rate of the second incision reaction, K_obs2, must be much greater than the rate of the first incision. b Effects of MgCl₂ on the kinetics of drUvrABC dual-incision activity. Time-course experiments following the accumulation of the 12 mer product (purple) and the intermediate products, 30 mer (orange), resulting from 3′ incision or 32 mer (red), resulting from 5′ incision, as a function of time. The graphs present the mean (filled triangles) and standard deviation of three individual replicates shown as open circles. Reactions were performed at 37 °C using 25 nM F26-seq1 substrate, 1 µM drUvrA1, 0.5 µM drUvrB, and 2 µM drUvrC in the presence of 1 mM (left), 2.5 mM (middle), or 10 mM (right) MgCl₂.

Fig. 5. Substrate specificity of drUvrABC.

Kinetics of release of the 12 mer product by drUvrABC from either fluorescein-conjugated DNA substrates (a), F26-seq1 (blue) or F26-seq2 (red), or biotin-conjugated substrates (b), B26-seq1 (green) or B26-strep-seq1 (plum), detailed in Supplementary Table 3. The observed rates of product release, K_obs, corresponding to the rates of the dual-incision reaction, were determined for each substrate by fitting the data points to either a single exponential model (in a) or a sigmoidal model (in b). a–b Reactions were performed at 37 °C using 25 nM DNA substrate, 1 µM drUvrA1, 0.5 µM drUvrB, and 2 µM drUvrC in the presence of 2.5 mM MgCl₂. Reactions were started with 2.5 mM ATP. The graphs present the mean (filled triangles) and standard deviation of at least three individual replicates shown as open circles.