Double-stranded gap repair in the photosynthetic prokaryote Synechococcus R2

Abstract

The photosynthetic cyanobacterium Synechococcus R2 is transformed by chimeric donor molecules lacking a functional replication origin but containing a region of homology to the recipient chromosome. These integrating donor molecules consist of a fragment of Synechococcus R2 chromosomal DNA cloned in the Escherichia coli vector pBR322 and interrupted by a piece of foreign DNA. During integration, this interrupting DNA is often lost by nonreciprocal exchange between homologous regions of donor and recipient. When transformed with donor molecules containing in vitro-generated double-stranded gaps or deletions as large as 20 kilobase pairs in the fragment homologous to the recipient chromosome, Synechococcus R2 can repair these lesions by using recipient information. Chromosomal DNA of the resulting transformants contains direct repeats of the recipient copy on either side of integrated pBR322 DNA. Homologous recombination between these repeats generates a circular molecule that can be recovered by transformation to E. coli. Plasmids recovered in E. coli contain the entire copy of information initially present in the region of the Synechococcus recipient corresponding to the donor gap or deletion. We suggest applications of this mechanism for cloning of genes identified by transposon mutagenesis.