Flow linear dichroism and electron microscopic analysis of protein-DNA complexes of a mutant UvrB protein that binds to but cannot kink DNA.
(A)BC excinuclease of Escherichia coli is the enzymatic activity resulting from sequential and partially overlapping actions of UvrA, UvrB, and UvrC protein. UvrA is a molecular matchmaker which promotes the formation of a stable UvrB-damaged DNA complex in which the DNA is kinked by about 130 degrees. The UvrB-DNA complex is then recognized by UvrC and two incisions are made in the DNA by the joint actions of UvrC and UvrB. A mutant of UvrB (D478A) can be loaded onto the DNA but it does not interact with UvrC to cause a nick 3' to the lesion. Based on the lack of a DNase-I-hypersensitive site in the footprint of the mutant, it was proposed that the lack of incision was due to the inability of the mutant UvrB to kink the DNA. In the current study we have investigated the interaction of the mutant UvrB with DNA using two biophysical methods, flow linear dichroism and electron microscopy. Both methods reveal that the mutant UvrB is unable to bend DNA.
Hsu
D S
DS
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
Takahashi
M
M
Delagoutte
E
E
Bertrand-Burggraf
E
E
Wang
Y H
YH
Norden
B
B
Fuchs
R P
RP
Griffith
J
J
Sancar
A
A
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects / Q000648:ultrastructure; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins; D008854:Microscopy, Electron / Q000379:methods; D009154:Mutation / Q000502:physiology*; D009690:Nucleic Acid Conformation; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D004247:DNA; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli; C036385:endodeoxyribonuclease uvrABC; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases