Analysis of the recE locus of Escherichia coli K-12 by use of polyclonal antibodies to exonuclease VIII.
Exonuclease VIII (exoVIII) of Escherichia coli has been purified from a strain carrying a plasmid-encoded recE gene by using a new procedure. This procedure yielded 30 times more protein per gram of cells, and the protein had a twofold higher specific activity than the enzyme purified by the previously published procedure (J. W. Joseph and R. Kolodner, J. Biol. Chem. 258:10411-10417, 1983). The sequence of the 12 N-terminal amino acids was also obtained and found to correspond to one of the open reading frames predicted from the nucleic acid sequence of the recE region of Rac (C. Chu, A. Templin, and A. J. Clark, manuscript in preparation). Polyclonal antibodies directed against purified exoVIII were also prepared. Cell-free extracts prepared from strains containing a wide range of chromosomal- or plasmid-encoded point, insertion, and deletion mutations which result in expression of exoVIII were examined by Western blot (immunoblot) analysis. This analysis showed that two point sbcA mutations (sbcA5 and sbcA23) and the sbc insertion mutations led to the synthesis of the 140-kilodalton (kDa) polypeptide of wild-type exoVIII. Plasmid-encoded partial deletion mutations of recE reduced the size of the cross-reacting protein(s) in direct proportion to the size of the deletion, even though exonuclease activity was still present. The analysis suggests that 39 kDa of the 140-kDa exoVIII subunit is all that is essential for exonuclease activity. One of the truncated but functional exonucleases (the pRAC3 exonuclease) has been purified and confirmed to be a 41-kDa polypeptide. The first 18 amino acids from the N terminus of the 41-kDa pRAC3 exonuclease were sequenced and fond to correspond to one of the translational start signals predicted from the nucleotide sequence of radC (Chu et al., in preparation).
Luisi-DeLuca
C
C
Laboratory of Molecular Genetics, Dana-Farber Cancer Institute, Boston, Massachusetts.
Clark
A J
AJ
Kolodner
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RD
D000483:Alleles; D000595:Amino Acid Sequence; D000906:Antibodies; D000936:Antigen-Antibody Complex / Q000032:analysis; D001483:Base Sequence; D015153:Blotting, Western; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D005796:Genes; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids
D000906:Antibodies; D000936:Antigen-Antibody Complex; D005090:Exodeoxyribonucleases; C026870:exodeoxyribonuclease VII