Fig. 2: Optimization of the in vitro NER system. | Communications Biology

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

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

Fig. 2

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.

Back to article page