Fig. 1: Deinococcus radiodurans UvrABC system. | Communications Biology

Fig. 1: Deinococcus radiodurans UvrABC system.

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

Fig. 1

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.

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