Electron microscopic study of (A)BC excinuclease. DNA is sharply bent in the UvrB-DNA complex.
Nucleotide excision repair in Escherichia coli is initiated by the UvrA, UvrB and UvrC proteins. UvrA is the damage recognition subunit, makes an A2B1 complex with the targeting subunit UvrB, and the complex binds to the lesion site; UvrA dissociates leaving behind a very stable UvrB-DNA complex that is recognized by the trigger subunit, UvrC, and the ensuing UvrB-UvrC heterodimer makes two incisions, one on either side of the lesion. Using electron microscopy, we investigated the structures of these early A, A-B intermediates on DNA containing ultraviolet light photoproducts. UvrA, which is known to bind to DNA as a dimer and produce a DNase I footprint of 33 base-pairs does not change the trajectory of DNA appreciably. The A2B1 complex clearly shows a bipartite structure and its effect on the trajectory of the DNA was not consistently straight or kinked. In contrast, the DNA in the preincision UvrB-DNA complex appears to be severely kinked; 43% of the molecules are bent by 80 degrees or more, with an average bending angle of 127 degrees. It appears that protein-induced bending is an important step on the pathway leading to excision of the damaged nucleotide by (A)BC excinuclease.
Shi
Q
Q
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
Thresher
R
R
Sancar
A
A
Griffith
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D004265:DNA Helicases; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism / Q000528:radiation effects / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000648:ultrastructure; D004706:Endodeoxyribonucleases / Q000648:ultrastructure*; D004926:Escherichia coli / Q000201:enzymology / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C058093:UvrB protein, E coli; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases