Abstract
The UvrB-DNA preincision complex plays a key role in nucleotide excision repair in Escherichia coli. To study the formation of this complex, derivatives of a DNA substrate containing a cholesterol adduct were constructed. Introduction 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 by 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. Similar modifications at the 5' side of the damage, however, gave different results. The introduction of a single strand nick at the 5' incision site completely abolished the UvrA-mediated formation of the UvrB-DNA complex. Deletion of both DNA strands up to the 5' incision site also prevented the UvrA-mediated loading of UvrB onto the damaged site, but UvrB by itself could bind very efficiently. This demonstrates that the UvrB protein is capable of recognizing damage without the matchmaker function of the UvrA protein. Our 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.
Highlights
The UvrB-DNA preincision complex plays a key role in nucleotide excision repair in Escherichia coli
Our results indicate that the UvrAmediated loading of the UvrB protein is an asymmetric process, which starts at the 5 side of the damage
Several experiments described in this paper are in agreement with such a mechanism of UvrB loading: (i) On a truncated DNA fragment, which has 7 base pairs at the 5Ј side of the damage, UvrA can no longer load the UvrB protein
Summary
The UvrB-DNA preincision complex plays a key role in nucleotide excision repair in Escherichia coli. We show that a doublestranded substrate in which DNA at the 3Ј side of the damage has been removed up to the 3Ј incision site can still form a stable preincision complex via the UvrA-mediated loading of the UvrB protein.
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