Occurrence of three-stranded DNA within a RecA protein filament.

A RecA protein-generated triple-stranded DNA species can be observed by electron microscopy, within narrowly defined conditions. Three-stranded DNA is detected only when initiation of normal DNA strand exchange is precluded by heterologous sequences within the duplex DNA substrate, when ATP is hydrolyzed, and when the DNA is cross-linked with a psoralen derivative prior to removal of RecA filaments. When adenosine 5'-O-(thiotriphosphate) is used, only the product hybrid duplex DNA can be cross-linked within the RecA filament. The third strand is either displaced or interwound in a conformation that does not permit cross-linking. When ATP is hydrolyzed by RecA, all three strands are cross-linked within the filament in a complex pattern that suggests a dynamic structure. This structure is altered when RecA protein is removed before cross-linking. Hsieh et al. (1990) and Rao et al. (1991, 1993) have proposed, on the basis of nuclease protection and chemical modification studies, that a stable triple-stranded DNA species can persist after removal of RecA protein. We have been unable to visualize these triple-stranded structures by the methods used in the present investigation. When RecA removal was followed immediately by interstrand cross-linking, only the two strands of the hybrid duplex DNA were cross-linked.