Three-stranded paranemic joints: architecture, topological constraints and movement

Abstract

The RecA and SSB proteins will catalyze the joining of two DNA molecules containing homologous sequences but lacking homologous ends in a reaction termed paranemic joining. The absence of homologous ends can be achieved by (1) pairing two circular DNAs or (2) using linear DNA(s) with ends lacking homology to the pairing partner. Here we have used electron microscopy (EM) to examine such pairings. Circular M13 single-stranded (ss) DNA enveloped by RecA protein into a presynaptic filament was paired with linear M13mp7 double-stranded (ds) DNA containing non-M13 sequences at its ends. Joint complexes were frequently seen in which the dsDNA was joined with the presynaptic filament over several kilobase (10(3) bases) lengths of the dsDNA. In this region, the presynaptic filament appeared disorganized as contrasted to the customary helical structure of the filament containing only a single strand of DNA. The same ultrastructure, but with greater detail, was observed when the samples were prepared for EM without fixation using a new method of fast-freezing and freeze-drying. EM immunogold staining demonstrated the presence of SSB protein in the disorganized region containing all three strands, but not in the regular helically arranged region. Psoralen photo-crosslinking of the DNA in the joint complexes revealed that the three DNA strands were in close proximity only over a single short (200 to 300 base-pairs) region. The joining of nicked circular M13 dsDNA and presynaptic filaments containing circular M13 ssDNA resulted in the intertwining of the dsDNA about the circular presynaptic filament. The joints produced in this case were short, as was the single region of psoralen photo-crosslinking of the three DNA strands. A model of how these long three-stranded joints form is presented involving the movement of a short "true" paranemic joint along the presynaptic filament.