Abstract
(A)BC excinuclease of Escherichia coli removes damaged nucleotides from DNA by hydrolyzing the 8th phosphodiester bond 5' and the 15th phosphodiester bond 3' to the modified base. The activity results from the ordered action of UvrA, UvrB, and UvrC proteins. The role of UvrA is to help assemble the UvrB.DNA complex, and it is not involved in the actual incision reactions which are carried out by UvrB and UvrC. To investigate the role of UvrC in the nuclease activity a subset of His, Asp, and Glu residues in the C-terminal half of the protein were mutagenized in vitro. The effect of these mutations on UV resistance in vivo and incision activity in vitro were investigated. Mutations, H538F, D399A, D438A, and D466A conferred extreme UV sensitivity. Enzyme reconstituted with these mutant proteins carried out normal 3' incision but was completely defective in 5' incision activity. Our data suggest that UvrC makes the 5' incision by employing a mechanism whereby the three carboxylates acting in concert with H538 and a Mg2+ ion facilitate nucleophilic attack by an active site water molecule.
Highlights
(A)BCexcinuclease of Escherichia coliremoves dam- base catalysis (Saenger, 1991)
Enzyme reconstituted with these We found that mutations in 4 residues H538, D399, D438, mutant proteins carriedout normal 3’ incision but was and D466 abolish the 5’ incision activity of (A)BC
Isolation and i n Vivo Characterization of uvrC Mutants-We havepreviously shown that thBeacillus subtilis uvrCgene (Chen et al, 1988) complements uvrC- mutations in E. coli (Lin and Sancar, 1990) and that the active site of UvrC is withinthecarboxyl-terminal half of theprotein(Linand
Summary
Were identified by single-stranded DNA sequencing, and the mutated genes were replaced the wild type uurC in pDR3274. Cells were collected by centrifugation, resuspended in 0.5 ml of buffer A, transferred to 1.5-ml microcentrifuge tubes, and frozen in a dry ice-ethanol bath. The supernatanwt as transferred into another tube, and pl of single-stranded DNA cellulose suspension in buffer B (-20-p1 packed volume) was added.The tubewas inverted. + resuspending the resin in 200 pl of buffer B 1.0 M KC1, and the only marginally overproduced and was not investigated in eluted protein was separated from the resin by centrifugation. The DNAwas collected by centrifugation, dried, resuspended in a formamide/dye mixture, and analyzed on an 8%polyacrylamide sequencing gel
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