OCCURRENCE OF 3-STRANDED DNA WITHIN A RECA PROTEIN FILAMENT

A RecA protein-generated triple-stranded DNA species can be observed b y electron microscopy, within narrowly defined conditions, Three stran ded DNA is detected only when initiation of normal DNA strand exchange is precluded by heterologous sequences within the duplex DNA substrat e, when ATP is hydrolyzed, and when the DNA is cross-linked with a pso ralen 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 d isplaced or interwound in a conformation that does not permit crosslin king, When ATP is hydrolyzed by RecA, all three strands are cross-link ed within the filament in a complex pattern that suggests a dynamic st ructure, This structure is altered when RecA protein is removed be for e cross-linking, Hsieh ct al, (1990) and Rao ct al, (1991, 1993) have proposed, on the basis of nuclease protection and chemical modificatio n studies, that a stable triple-stranded DNA species can persist after removal of RecA protein, We have been unable to visualize these tripl e-stranded structures by the methods used in the present investigation , When RecA removal was followed immediately by interstrand cross-link ing, only the two strands of the hybrid duplex DNA were cross linked.