RECOMBINATION-DEPENDENT REPAIR OF DNA DOUBLE-STRAND BREAKS WITH PURIFIED PROTEINS FROM ESCHERICHIA-COLI

We have developed an in vitro system in which repair of DNA double-str and breaks is performed by purified proteins of Escherichia coil, A se gment was deleted from a circular duplex DNA molecule by restriction a t two sites, 3' single-stranded overhangs were introduced at both ends of the remaining linear fragment, In a first step, RecA protein catal yzed the formation of a D-loop between one single-stranded tail and a homologous undeleted supercoiled DNA molecule, In a second step, E. co il DNA polymerase II or III used the 3' end in the D-loop as a primer to copy the missing sequences of the linear substrate on one strand of the supercoiled template, Under proper conditions, the integrity of t he deleted substrate was restored, as shown by analysis of the product s by electrophoresis, restriction, and transformation, In this reactio n, DNA synthesis is strictly dependent on recombination, and repair is achieved without formation of a Holliday junction.