The effect of the DNA flanking the lesion on formation of the UvrB-DNA preincision complex - Mechanism for the UvrA-mediated loading of UvrB onto a DNA damaged site

The UvrB-DNA preincision complex plays a key role in nucleotide excision re pair in Escherichia coli. To study the formation of this complex, derivativ es of a DNA substrate containing a cholesterol adduct were constructed. Int roduction of a single strand nick into either the top or the bottom strand at the 3' side of the adduct stabilized the UvrB-DNA complex, most Likely b y the release of local stress in the DNA Removal of both DNA strands up to the 3' incision site still allowed formation of the preincision complex. Si milar modifications at the 5' side of the damage, however, gave different r esults. The introduction of a single strand nick at the 5' incision site co mpletely abolished the UvrA-mediated formation of the UvrB-DNA complex. Del etion of both DNA strands up to the 5' incision site also prevented the Uvr A-mediated loading of UvrB onto the damaged site, but UvrB by itself could bind very efficiently, This demonstrates that the UvrB protein is capable o f recognizing damage without the matchmaker function of the UvrA protein. O ur results also indicate that the UvrA-mediated loading of the UvrB protein is an asymmetric process, which. starts at the 5' side of the damage.