DNA helicase I, the traI gene product of the Escherichia coli F factor, was shown to be associated with endonuclease activity specific for the transfer origin of the F plasmid, oriT. In the presence of Mg2+, the purified enzyme forms a complex, stable in the presence of sodium dodecylsulfate (SDS) with a negatively superhelical chimeric plasmid containing oriT. The enzyme nicks and, after this, apparently binds to the 5′ nick terminus when this complex is heated in the presence of SDS and/or EDTA or treated with proteinase K. Dideoxy sequencing locates the nick site in the F DNA strand transferred during bacterial conjugation after nucleotide 138 clockwise of the mid‐point of the BglII site at 66.7 kb of the F genetic map. A sequencing stop after nucleotide 137 of this strand (where oriT‐nicking seems to occur in vivo) is possibly an artefact caused by helicase I protein attached to the 5′ terminal nucleotide. Deletion in the amino‐terminal part of the traI polypeptide abolishes the oriT‐nicking activity while leaving the strand‐separating activity intact. These results confirm the prediction from genetic studies that helicase I is bifunctional with site‐specific endonuclease and strand‐separating activities.
Ether-permeabilized (nucleotide-permeable) cells of Escherichia coli show excision repair of their DNA after having been exposed to the carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr) and methyl methanesulfonate (MeS0,OMe) which are known to bind covalently to DNA. Defect mutations in genes uvrA, uvrB, uvrC, r e d , recB, recC and rep did not inhibit this excision repair.Enzymic activities involved in this repair were identified by measuring size reduction of DNA, DNA degradation to acid-soluble nucleotides and repair polymerization.1. In permeabilized cells methyl and ethyl nitrosourea induced endonucleolytic cleavage of endogenous DNA, as determined by size reduction of denatured DNA in neutral and alkaline sucrose gradients. An enzymic activity from E. coli K-12 cell extracts was purified (greater than 2000-fold) and was found to cleave preferentially methyl-nitrosourea-treated DNA and to convert the methylated supercoiled DNA duplex (RF I) of phage @X 174 into the nicked circular form.2. Degradation of alkylated cellular DNA to acid solubility was diminished in a mutant lacking the 5'+3' exonucleolytic activity of DNA polymerase I but was not affected in a mutant which lacked the DNA polymerizing but retained the 5'+3' exonucleolytic activity of DNA polymerase I.3. An easily measurable effect is carcinogen-induced repair polymerization, making it suitable for detection of covalent binding of carcinogens and potentially carcinogenic compounds.Excision repair plays an important role in the removal of chemically altered bases from DNA [l -61. Ultraviolet light-induced repair in Escherichia coli involves an endonuclease activity [7] which hydrolyzes a phosphodiester bond close to the pyrimidine dimer, an exonuclease activity [8,9] which extends the nick by removing the dimer, a DNA polymerase, generally polymerase I [lo, 111, which fills the gap using the opposite strand as template and polynucleotide ligase [12] which joins the free ends. Although the funda-
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