The C-terminal region of the UvrB protein of Escherichia coli contains an important determinant for UvrC binding to the preincision complex but not the catalytic site for 3'-incision.
The UvrABC endonuclease from Escherichia coli repairs damage in the DNA by dual incision of the damaged strand on both sides of the lesion. The incisions are in an ordered fashion, first on the 3'-side and next on the 5'-side of the damage, and they are the result of binding of UvrC to the UvrB-DNA preincision complex. In this paper, we show that at least the C-terminal 24 amino acids of UvrB are involved in interaction with UvrC and that this binding is important for the 3'-incision. The C-terminal region of UvrB, which shows homology with a domain of the UvrC protein, is part of a region that is predicted to be able to form a coiled-coil. We therefore propose that UvrB and UvrC interact through the formation of such a structure. The C-terminal region of UvrB only interacts with UvrC when present in the preincision complex, indicating that the conformational change in UvrB accompanying the formation of this complex exposes the UvrC binding domain. Binding of UvrC to the C-terminal region of UvrB is not important for the 5'-incision, suggesting that for this incision a different interaction of UvrC with the UvrB-DNA complex is required. Truncated UvrB mutants that lack up to 99 amino acids from the C terminus still give rise to significant incision (1-2%), indicating that this C-terminal region of UvrB does not participate in the formation of the active site for 3'-incision. This region, however, contains the residue (Glu-640) that was proposed to be involved in 3'-catalysis, since a mutation of the residue (E640A) fails to promote 3'-incision (Lin, J.J., Phillips, A.M., Hearst, J.E., and Sancar, A. (1992) J. Biol. Chem. 267, 17693-17700). We have isolated a mutant UvrB with the same E640A substitution, but this protein shows normal UvrC binding and incision in vitro and also results in normal survival after UV irradiation in vivo. As a consequence of these results, it is still an open question as to whether the catalytic site for 3'-incision is located in UvrB, in UvrC, or is formed by both proteins.
Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
G F
GF
Moolenaar
K L
KL
Franken
D M
DM
Dijkstra
J E
JE
Thomas-Oates
R
R
Visse
P
P
van de Putte
N
N
Goosen
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001419:Bacteria / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004249:DNA Damage; D004265:DNA Helicases; D017931:DNA Primers; D004260:DNA Repair; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C058093:UvrB protein, E coli; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli; C036385:endodeoxyribonuclease uvrABC; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases