Characterization of the Escherichia coli damage-independent UvrBC endonuclease activity

Incision of damaged DNA templates by UvrBC in Escherichia coli depends on U vrA, which loads UvrB on the site of the damage. A 50-base pair 3' prenicke d DNA substrate containing a cholesterol lesion is incised by UvrABC at two positions 5' to the lesion, the first incision at the eighth and the secon d at the 15th phosphodiester bond, Analysis of a 5' prenicked cholesterol s ubstrate revealed that the second 5' incision is efficiently produced by Uv rBC independent of UvrA This UvrBC incision was also found on the same subs trate without a lesion and, with an even higher efficiency, on a DNA substr ate containing a 5' single strand overhang. Incision occurred in the presen ce of ATP or ADP but not in the absence of cofactor. We could show an inter action between UvrB and UvrC in solution and subsequent binding of this com plex to the substrate with a 5' single strand overhang. Analysis of mutant UvrB and UvrC proteins revealed that the damage-independent nuclease activi ty requires the protein-protein interaction do; mains, which are exclusivel y needed for the 3' incision on damaged substrates, However, the UvrBC inci sion uses the catalytic site in UvrC which makes the 5' incision on damaged DNA substrates.