Analysis of the recE locus of Escherichia coli K-12 by use of polyclonal antibodies to exonuclease VIII

Abstract

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 found to correspond to one of the translational start signals predicted from the nucleotide sequence of racC (Chu et al., in preparation).sbcA mutations suppress the conjugation-mediated recombination deficiency, UV sensitivity, and mitomycin C sensitivity of Escherichia coli strains containing recB or recC mutations or both (3). In addition, an sbcA mutation in a recB recC mutant background stimulates the recombination of circular monomer and dimer and linear dimer plasmid substrates 20-to 100-fold and increases the efficiency of recombination-mediated transformation of linear dimer plasmids at least 100-fold (10,19,26,31).One additional phenotype associated with the presence of an sbcA mutation is the presence of an ATP-independent DNase, exonuclease VIII (exoVIII;3,25). ExoVIII is a highly processive 5'-to-3' exonuclease with a subunit molecular weight of 140,000 (21, 25). The enzyme preferentially degrades double-stranded linear DNA and has no activity on single-stranded DNA or double-stranded circular DNA in a supercoiled, nicked, or gapped configuration (21). ExoVIII is functionally similar to A exonuclease (6,21,22,25,27), and can substitute for the X red functions (12). The structural gene for exoVIII has been termed recE (25). recE mutations reduce elevated levels of conjugational and plasmid recombination and UV and mitomycin C resistance produ...

Identification and characterization of the Escherichia coli RecT protein, a protein encoded by the recE region that promotes renaturation of homologous single-stranded DNA

“…pRac3 exo was purified to homogeneity by precipitation with ammonium sulfate and chromatography on PBE94 ( Fig. 1 and Table 1) by a modification of a previously published procedure (31). Analysis by NaDodSO4-PAGE indicated that the final fraction was greater than 99% pure (Fig.…”

“…The sizes of the pRac3 exo, p33, and exoVIII polypeptides were estimated by NaDodSO4-PAGE to be 42.5, 32.5, and 140 kDa, respectively (8,31; this work). exoVIII appears to migrate anomalously during analysis by NaDodSO4-PAGE, since the recE and recT DNA sequences predict that exoVIII should be a 126-kDa protein (4,9).…”

“…Genetic and biochemical analyses have demonstrated that the E. coli recE region encodes a 140,000-molecular-weight exonuclease, called exoVIII, that is required for recombination in recB recC sbcA mutants (4,9,13,19,20,22,31,32,49,54). This exonuclease can be truncated at its amino terminus and still retain enzymatic and biological activities (4,8,31,55).…”

“…This exonuclease can be truncated at its amino terminus and still retain enzymatic and biological activities (4,8,31,55). Clark and colleagues (9) have demonstrated that the recE region contains two overlapping reading frames, now called recE and recT, capable of encoding 96.2-and 29.5-kDa proteins, respectively.…”

“…Purification of pRac3 exo was done by a modification of a previously published procedure (31) and monitored by both NaDodSO4-PAGE and exonuclease assays. p33 purification was monitored by NaDodSO4-PAGE.…”