PURIFICATION AND SUBUNIT ANALYSIS OF EXONUCLEASE VII OF E. COLI; ISOLATION OF NEGATIVE-XSEB STRAINS AND LAMBDA-DXSEA TRANSDUCING PHAGE

Abstract

Exonuclease VII, isolated from Escherichia coli, has been purified to 87% homogeneity. The protein is composed of two non-identical subunits of 54,000 and 10,500 molecular weights as shown by restoration of exonuclease VII activity upon renaturation of both these polypeptides isolated from an SDS-polyacrylamide gel. The 10,500 and 54,000 dalton proteins are present in a four-to-one molar ratio respectively.;The isolation of E. coli strains containing deletions from guaB through xseA and from guaB through upp loci is described. These strains were derived by temperature induction of (lamda)cI857 prophage integrated in the guaB locus of strain KS504. This strain was also utilized in the isolation of (lamda)dxseA transducing phage which were found to restore exonuclease VII activity in strain KLC381 (del(guaB-xseA)) but did not produce increased quantities of exonuclease VII activity in xseA('+) strains upon infection or lysogenization.;Seven new mutant strains of E. coli containing deficiencies in exonuclease VII activity are described. Four of these strains contain lesions in the previously identified structural gene for the enzyme, xseA. However, the other three strains contain mutations in a previously unidentified locus, designated xseB. This gene lies between proC and acrA loci and is a structural gene for exonuclease VII as determined by analysis of strain KLC835 (xseB3) which contains temperature sensitive exonuclease VII activity. In addition, mixing and renaturation of the subunits of exonuclease VII isolated from strains HMS137 (xse('+)) and KLC835 (xseB3) demonstrate that xseB encodes the 10,500 dalton subunit of the enzyme.