PRIMARY PRODUCTS OF BREAK-INDUCED RECOMBINATION BY ESCHERICHIA-COLI RECE PATHWAY

Alternative models for break-induced recombination predict different d istributions of primary products. The double-stranded break-repair mod el predicts a noncrossover product and equimolar amounts of two crosso ver products. The one-end pairing model predicts two crossover product s, but not necessarily in equimolar amounts, and the single-stranded a nnealing model predicts deletion of the fragment between the pairing s equences. Depending on the structure of the recombining substrate(s) a nd the nature of the resectioning step that precedes strand annealing, the single-stranded annealing mechanism would yield only one or both crossover products. We tested these predictions for the RecE recombina tion pathway of Escherichia coli. Nonreplicating intramolecular recomb ination substrates with a double-stranded break (DSB) within one copy of a direct repeat were released from chimera lambda phage by in vivo restriction, and the distribution of primary circular recombination pr oducts was determined. Noncrossover products were barely detectable, a nd the molar ratio of the two crossover products was proportional to t he length ratio of the homologous ends flanking the DSB. These results suggest an independent pairing of each end with the intact homolog an d argue against the double-stranded break-repair model. However, the r esults do not distinguish alternative pairing mechanisms (strand invas ion and strand annealing). The kinetics of heteroduplex formation and heteroduplex strand polarity were investigated. Immediately following the DSB induction, heteroduplex formation was done by pairing the stra nds ending 3' at the break A slow accumulation of the complementary he teroduplex made by the pairing of the strands ending 5' at the break ( 5' heteroduplexes) was observed at a later stage. The observed bias in heteroduplex strand polarity depended on DSB induction at a specific site. The 5' heteroduplexes may have been generated by reciprocal stra nd exchange, pairing that is not strand specific, or strand-specific p airing induced at random breaks.