Uptake of homologous single-stranded fragments by superhelical DNA: III. The product and its enzymic conversion to a recombinant molecule

Abstract

When superhelical DNA (RFI) † † Terminology and abbreviations: the closed double-stranded circular form of phage φX174 or G4 DNA is termed RFI or form I; circular duplexes containing one or more interruptions are termed form II. The molecule formed by the uptake of a homologous single-stranded fragment by superhelical DNA is called a complex. The associated single strand is sometimes designated as the third strand or donor strand. As denned by Kasamatsu et al. (1971) the term displacement loop or D-loop is used to describe a particular kind of triple-stranded region in DNA containing 2 paired strands and an unpaired strand, the latter covalently linked at both ends to the rest of the duplex DNA. The symbols l, r, and r c stand, respectively, for the number of nucleotides in a fragment, the number of residues of fragment nucleotide per molecule of circular DNA, and the number of residues of fragment nucleotide per molecule of complex. A bar above the symbol indicates a mean value. Concentrations of DNA are given as moles of phosphorus except as indicated. of phages φX174 or G4 takes up a homologous single-stranded fragment, RF DNA and fragment are linked by as many as 300 base-pairs, and a corresponding length of one strand of the RFI is displaced, forming a displacement loop (D-loop). The length of the base-paired region was estimated from the fraction of the associated 32P-labeled fragment that was resistant to digestion by exonuclease VII, as well as by electron microscopy. Dissociation of the fragment by heating was characterized by a sharp melting curve. The displaced strand of the RF DNA was digested by two endonucleases that act on single-stranded DNA, the S 1 nuclease of Aspergillus oryzae and the recBC DNAase of Escherichia coli. Acting on complexes, both enzymes converted the form I [ 3H]DNA into form II DNA, and left some of the associated 32P-labeled fragment undigested. The remaining 32P-labeled fragment could no longer be displaced by branch migration, as expected if the displaced strand of the RF DNA were digested. The action of S 1 nuclease also produced the amount of acid-soluble 3H expected from digestion of the D-loop. Treatment of such digested complexes with polynucleotide ligase covalently linked about 35% of the remaining 32P-labeled fragment to 3H-labeled strands, which proves that S 1 nuclease digested the D-loop.