Inserting Site-Specific DNA Lesions into Whole Genomes

Abstract

Here, we describe a methodology that allows the insertion of site-specific DNA lesions into genomes in living cells. The technique involves the integration of a plasmid containing a site-specific lesion engineered in vitro into a precise location in the genome via the site-specific recombination reaction from phage lambda. The notion of DNA lesion is not restricted to chemically modified nucleotides but also refers to unusual DNA structures. This method will be instrumental to study qualitatively and quantitatively the genetic consequences of site-specific lesions in vivo; moreover, it does also allow analyzing the molecular structure of stalled replication forks at well-defined locations.

Keywords: DNA adducts; DNA damage tolerance; Damage avoidance; Homology-directed recombination; Site-specific recombination; Translesion synthesis; Unusual DNA structures.