Characterization of the Escherichia coli damage-independent UvrBC endonuclease activity.
Incision of damaged DNA templates by UvrBC in Escherichia coli depends on UvrA, which loads UvrB on the site of the damage. A 50-base pair 3' prenicked DNA substrate containing a cholesterol lesion is incised by UvrABC at two positions 5' to the lesion, the first incision at the eighth and the second at the 15th phosphodiester bond. Analysis of a 5' prenicked cholesterol substrate revealed that the second 5' incision is efficiently produced by UvrBC independent of UvrA. This UvrBC incision was also found on the same substrate without a lesion and, with an even higher efficiency, on a DNA substrate containing a 5' single strand overhang. Incision occurred in the presence of ATP or ADP but not in the absence of cofactor. We could show an interaction between UvrB and UvrC in solution and subsequent binding of this complex to the substrate with a 5' single strand overhang. Analysis of mutant UvrB and UvrC proteins revealed that the damage-independent nuclease activity requires the protein-protein interaction domains, which are exclusively needed for the 3' incision on damaged substrates. However, the UvrBC incision uses the catalytic site in UvrC which makes the 5' incision on damaged DNA substrates.
Moolenaar
G F
GF
Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands.
Bazuine
M
M
van Knippenberg
I C
IC
Visse
R
R
Goosen
N
N
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004249:DNA Damage; D004265:DNA Helicases; D004706:Endodeoxyribonucleases; D004720:Endonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D013379:Substrate Specificity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C066567:UvrC protein, E coli; D004265:DNA Helicases