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 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.
Moolenaar
G F
GF
Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Monaco
V
V
van der Marel
G A
GA
van Boom
J H
JH
Visse
R
R
Goosen
N
N
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D002784:Cholesterol / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding
D001426:Bacterial Proteins; D018736:DNA Adducts; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D002784:Cholesterol; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli; C036385:endodeoxyribonuclease uvrABC; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases