Figure 6.
Characterization of the DNA cleavage reaction. The cloned-gene product purified from E. coli cells was used. (A) Inhibition by NaCN and KBH4. Each reaction contained 0.3 μg of photosensitization-treated pUC18 DNA, 10 ng of the enzyme, and indicated concentration of the inhibitor. Lanes 1 and 11, substrate alone; lane 2, without inhibitor; lanes 3–6, with NaCN; lanes 7–10, with KBH4. oc, open circles; cc, closed circles. (B) The labeled strand of the 50-bp synthetic substrate. Overline, dithymidylate; downward arrow, cleavage site for NlaIII and NspI; upward arrow, cleavage site for CPyD–DNA glycosylase/abasic lyase. (C) Cleavage of photosensitization-treated (lanes 1 and 2) or untreated (lanes 3 and 4) 50-bp substrate. Enzymes used: none (lane 1), 240 ng of the cloned-gene product (lane 2), 4 units of NlaIII (lane 3), 10 units of NspI (lane 4). M, oligonucleotide size markers (Pharmacia) labeled in the same way as the substrate; figures, chain lengths. In addition to the full-length 50-mer (arrowhead), the substrate preparation contained significant amounts of shorter, mostly 49-mer, contaminants.