The C-terminal half of UvrC protein is sufficient to reconstitute (A)BC excinuclease.
The UvrC protein is one of three subunits of the Escherichia coli repair enzyme (A)BC excinuclease. This subunit is thought to have at least one of the active sites for nucleophilic attack on the phosphodiester bonds of damaged DNA. To localize the active site, mutant UvrC proteins were constructed by linker-scanning and deletion mutagenesis. In vivo studies revealed that the C-terminal 314 amino acids of the 610-amino acid UvrC protein were sufficient to confer UV resistance to cells lacking the uvrC gene. The portion of the uvrC gene encoding the C-terminal half of the protein was fused to the 3' end of the E. coli malE gene (which encodes maltose binding protein), and the fusion protein MBP-C314C was purified and characterized. The fusion protein, in combination with UvrA and UvrB subunits, reconstituted the excinuclease activity that incised the eighth phosphodiester bond 5' and the fourth phosphodiester bond 3' to a psoralen-thymine adduct. These results suggest that the C-terminal 314 amino acids of UvrC constitute a functional domain capable of interacting with the UvrB-damaged DNA complex and of inducing the two phosphodiester bond incisions characteristic of (A)BC excinuclease.
Lin
J J
JJ
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
Sancar
A
A
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002872:Chromosome Deletion; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D015345:Oligonucleotide Probes; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli; C036385:endodeoxyribonuclease uvrABC