SEQUENCE-SPECIFIC INTERACTIONS OF UVRABC ENDONUCLEASE WITH PSORALEN INTERSTRAND CROSS-LINKS

The nature of the Uvr protein-DNA complexes formed on psoralen-DNA int erstrand cross-links was analyzed by DNase I footprinting and correlat ed with the incision efficiency of the UvrABC endonuclease on the cros s-links of different DNA sequences. Our results indicate that the repa ir specificity is dependent on the DNA sequence and the psoralen orien tation in the cross-link. On the strand that will be cut, a 30-nucleot ide long UvrAB footprint with a DNase I hypersensitive site at the 11t h nucleotide 5' to the lesion was observed and subsequently rearranged to a 22-nucleotide long UvrB-lesion footprint. On the strand that wil l not be cut, the UvrAB- lesion footprint had no 5' DNase I hypersensi tive site and did not form the UvrB-lesion footprint. Although UvrABC incision requires the formation of UvrB-lesion complex on the strand w hich will be cut, the affinities of these complexes do not correlate w ith the incision efficiencies, suggesting that the overall reaction ca n be driven forward by a favorable next step such as UvrC incision. A study of the time-dependent interconversion of UvrAB-lesion complex to UvrB-lesion complex on a cross-link revealed a secondary recognition of the UvrB-lesion complex by UvrA2(B) proteins in vitro.