Jul 4, 1997

Recombination-dependent repair of DNA double-strand breaks with purified proteins from Escherichia coli

The Journal of Biological Chemistry
P MorelE Cassuto

Abstract

We have developed an in vitro system in which repair of DNA double-strand breaks is performed by purified proteins of Escherichia coli. A segment was deleted from a circular duplex DNA molecule by restriction at two sites. 3' single-stranded overhangs were introduced at both ends of the remaining linear fragment. In a first step, RecA protein catalyzed the formation of a D-loop between one single-stranded tail and a homologous undeleted supercoiled DNA molecule. In a second step, E. coli 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 the deleted substrate was restored, as shown by analysis of the products by electrophoresis, restriction, and transformation. In this reaction, DNA synthesis is strictly dependent on recombination, and repair is achieved without formation of a Holliday junction.

  • References30
  • Citations4

Mentioned in this Paper

Bacterial Proteins
Rec A Recombinases
Alkalescens-Dispar Group
DNA, Viral
Base Excision Repair
Recombination, Genetic
RAD51
Oligonucleotide Primers
Electron Microscopy
DNA Polymerase III

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