INTERACTIONS OF 3 STRANDS IN JOINTS MADE BY RECA PROTEIN

RecA protein from Escherichia coli has been used to form a triple-stra nded DNA structure from either single-stranded M13 DNA or a single-str anded oligonucleotide plus a duplex oligonucleotide with a hairpin loo p. The secondary structure of purified deproteinized triplex was exami ned by probing with DNase I, P1 nuclease, potassium permanganate, and diethyl pyrocarbonate. The two strands destined to form heteroduplex D NA showed the same patterns of chemical modification and enzymatic dig estion as control duplex DNA, indicating that they formed a normal dup lex substructure. However, the nascent outgoing strand showed properti es consistent with a novel triplex structure: most of its purine resid ues, especially adenines, were hyperreactive to all probes. The patter ns of digestion by DNase I and P1 nuclease indicated that the nascent outgoing strand was not a freely mobile or single-stranded branch but rather was still interacting with the newly formed heteroduplex DNA. O n the basis of the planar base triads proposed previously (Rao et al., 1993) and energy minimization of a third strand in the major groove o f B-form DNA, we derived a model that helps to rationalize the propert ies revealed by chemical and enzymatic probing.