Figure 3. Time course of UvrABC^Tma and UvrABC^Bca incisions of trans- and cis-BPDE adducted oligonucelotide substrates. A. 60 °C B. 37 °C.

Oligonucleotides containing either a cis-, a trans- or no BPDE-adduct were site specifically labeled with [³²P] internally 19 bp from 3′-end to monitor the 5′ incision (producing a 32-mer fragment) for UvrABC^Bca and 3′ incision (producing a 20-mer fragment) for UvrABC^Tma, labeled 50-bp oligonucleotides were used as substrates in UvrABC reactions. UvrABC reactions were performed with twenty femto moles of substrates. Preincubation of substrates with UvrA 10 nM and UvrB 250 nM for 30 minutes at 60 °C, then add UvrC (100 nM) with incubation at varied time as indicated in the figure. Reaction products were denatured and resolved on 12% polyacrylamide sequencing gel. The intensities of the resolved bands were quantitated and the average incision ratio was calculated. Means ± SD from triplicate reactions are plotted.