THE ROLE OF NEGATIVE SUPERHELICITY AND LENGTH OF HOMOLOGY IN THE FORMATION OF PARANEMIC JOINTS PROMOTED BY RECA PROTEIN

Escherichia coli RecA protein pairs homologous DNA molecules to form p aranemic joints when there is an absence of a free end in the region o f homologous contact. Paranemic joints are a key intermediate in homol ogous recombination and are important in understanding the mechanism f or a search of homology. The efficiency of paranemic joint formation d epended on the length of homology and the topological forms of the dup lex DNA. The presence of negative superhelicity increased the pairing efficiency and reduced the minimal length of homology required for par anemic joint formation. Negative superhelicity stimulated joint format ion by favoring the initial unwinding of duplex DNA that occurred duri ng the homology search and was not essential in the maintenance of the paired structure. Regardless of length of homology, formation of para nemic joints using circular duplex DNA required the presence of more t han six negative supercoils. Above six negative turns, an increasing d egree of negative superhelicity resulted in a linear increase in the p airing efficiency. These results support a model of two distinct kinds of DNA unwinding occurring in paranemic joint formation: an initial u nwinding caused by heterologous contacts during synapsis and a later o ne during pairing of the homologous molecules.