Formation of linear plasmid multimers promoted by the phage lambda Red-system in lon mutants of Escherichia coli.
We report here the formation of plasmid linear multimers promoted by the Red-system of phage lambda using a multicopy plasmid comprised of lambda red alpha and red beta genes, under the control of the lambda cI857 repressor. Our observations have revealed that the multimerization of plasmid DNA is dependent on the red beta and recA genes, suggesting a concerted role for these functions in the formation of plasmid multimers. The formation of multimers occurred in a recBCD+ sbcB+ xthA+ lon genetic background at a higher frequency than in the isogenic lon+ host cells. The multimers comprised tandem repeats of monomer plasmid DNA. Treatment of purified plasmid DNA with exonuclease III revealed the presence of free double-chain ends in the molecules. Determination of the size of multimeric DNA, by pulse field gel electrophoresis, revealed that the bulk of the DNA was in the range 50-240 kb, representing approximately 5-24 unit lengths of monomeric plasmid DNA. We provide a conceptual framework for Red-system-promoted formation and enhanced accumulation of plasmid linear multimers in lon mutants of E. coli.
Mythili
E
E
Department of Biochemistry, Indian Institute of Science, Bangalore.
Muniyappa
K
K
D049069:ATP-Dependent Proteases; D001426:Bacterial Proteins / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics* / Q000502:physiology; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D049070:Protease La; D011938:Rec A Recombinases / Q000235:genetics; D011995:Recombination, Genetic; D012697:Serine Endopeptidases / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; C489340:recF protein, E coli; C066218:recF protein, Bacteria; D011938:Rec A Recombinases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La