Reconstitution of nucleotide excision nuclease with UvrA and UvrB proteins from Escherichia coli and UvrC protein from Bacillus subtilis.
Recently, an open reading frame which has a deduced amino acid sequence that shows 38% homology to Escherichia coli UvrC protein was found upstream of the aspartokinase II gene (ask) in Bacillus subtilis (Chen, N.-Y., Zhang, J.-J., and Paulus, H. (1989) J. Gen. Microbiol. 135, 2931-2940). We found that plasmids containing this open reading frame complement the uvrC mutations in E. coli. We joined the open reading frame to a tac promoter to amplify the gene product in E. coli and purified the protein to near homogeneity. The apparent molecular weight of the gene product is 69,000, which is consistent with the calculated molecular weight of 69,378 fro the deduced gene product of the open reading frame. The purified gene product causes the nicking of DNA at the 8th phosphodiester bond 5' and the 5th phosphodiester bond 3' to a thymine dimer when mixed with E. coli UvrA and UvrB proteins and a DNA substrate containing a uniquely located thymine dimer. We conclude that the gene product of the open reading frame is the B. subtilis UvrC protein. Our results suggest that the B. subtilis nucleotide excision repair system is quite similar to that of E. coli. Furthermore, complementation of the UvrA and UvrB proteins from a Gram-negative bacterium with the UvrC protein of Gram-positive B. subtilis indicates a significant evolutionary conservation of the nucleotide excision repair system.
Lin
J J
JJ
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
Sancar
A
A
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004265:DNA Helicases; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D015183:Restriction Mapping; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli; C036385:endodeoxyribonuclease uvrABC; D004265:DNA Helicases