Recombinant DNA molecules of bacteriophage phi chi174.

Abstract

ABSTRACT4 X174 DNA structures containing two different parental genomes were detected genetically and examined by electron microscopy. These structures consisted of two monomeric double-stranded DNA molecules linked in a figure 8 configuration. Such DNA structures were observed to be formed preferentially in host recA+ cells or recA + cell-free systems. Since the host recA + allele is required for most 4OX174 recombinant formation, we conclude that the observed figure 8 molecules are intermediates in, or end products of, a 4X174 recombination event.We propose that recombinant figure 8 DNA molecules arise as a result of "single-strand aggression," are stabilized by double-strand "branch migration," and represent a specific example of a common intermediate in genetic recombination.Genetic recombination in bacteriophage OX174 (1,2) and in the closely related bacteriophage S13 (3) has been analyzed extensively by both genetic (4-9) and physical (10-16) methods. Most OX174 recombinants are formed by a major pathway, Tessman's primary mechanism (4, 5), which requires the host recA + allele (6) but apparently does not need any of the nine known OX174 gene products (7). Recombinant formation via this major pathway involves two parental replicative form (RF) DNA molecules (5, 7), and occurs very early in the infection process (10). Single recombination events via the major pathway usually generate only one parental genotype and one recombinant (7). The goal of this work was to identify OX174 DNA structures which were formed by this major pathway.To determine whether a particular DNA structure was recombinant, parental replicative form DNA molecules were isolated from cells infected with two parental genotypes (11). After purification and fractionation of the DNA molecules by velocity and equilibrium sedimentation procedures (12, 13), the frequency of recombinants associated with various DNA structures was examined genetically using a spheroplast assay in which further recombination could not occur (10). In addition, we were able to identify putative recombinant DNA molecules formed in mixed infections by using two parental genotypes which could be distinguished by electron microscopy (13).In this paper we present electron micrographs of OX174 DNA molecules which apparently are recombinant. These structures appear to be "figure 8" molecules (15,17) We propose a simple mechanism to generate figure 8 DNA molecules that are recombinant. It is based on two molecular processes-"single-strand aggression" (11) and "branch migration" (19-21). These were previously implicated in recombinant formation in OX174 (10,11,13,15) and in other organisms (20,22). MATERIALS AND METHODSReplicative form and multiple length DNA molecules were isolated from cells infected with two genotypes (9, 11, 13, 23; R. M. Benbow, M. Eisenberg, and R. L. Sinsheimer, to be published). The fractionated DNA molecules were examined for recombinants by carrying out spheroplast infections (24), after which the progeny phage were assayed for wild-type recombina...

Genetic recombination in avian retroviruses

“…One of them, the DNA exchange model [3], was proposed at the time when it first seemed clear that circular DNA molecules were intermediates in the formation of proviral DNA. This, and an awareness of the discontinuity of the positive strands, made plausible a type of exchange that had been suggested earlier for the circular DNA of bacteriophage 4x174 [23]. It can be seen that this model is similar in concept, though different in detail, to the displacement/ assimilation model.…”

Uptake of homologous single-stranded fragments by superhelical DNA: a possible mechanism for initiation of genetic recombination.

“…We were led to do these experiments by speculations about the initiation of general genetic recombination (1,24). According to one version of such speculations, an early event in recombination is the uptake by a recipient double-stranded molecule of a redundant strand displaced from a homologous donor.…”

ATP-dependent renaturation of DNA catalyzed by the recA protein of Escherichia coli.

“…This process, involving transfer of homologous regions of DNA, depends upon the annealing of complementary strands to produce, at an early stage in recombination, a region of duplex DNA containing one strand from each parental DNA molecule (3,4). Numerous studies have suggested that such heteroduplex overlaps are not formed in recA mutants, in contrast to other recombination-deficient mutants (e.g., recB, recC) (5)(6)(7)(8)(9)(10). Thus, although a number of genes can affect recombination, only the recA function has been implicated in strand transfer.…”