Role of DNA ligase in the illegitimate recombination that generates lambdabio-transducing phages in Escherichia coli

We studied the role of DNA ligase in illegitimate recombination in Escheric hia coli. A temperature-sensitive mutation in the lig gene reduced the freq uency with which lambda bio-transducing phages were generated to 10-14% of that of wild type under W irradiation. Reintroduction of the lig gene into this mutant restored tire frequency of recombinant phage generation to that of wild type. Furthermore, overexpression of DNA ligase enhanced illegitim ate recombination by 10-fold with or without W irradiation. In addition, wh en DNA ligase was present in only limited amounts, UV-induced or spontaneou s illegitimate recombination occurred exclusively at hotspot sites that hav e relatively long sequences of homology (9 or 13 bp). However, when DNA lig ase was overexpressed, most of the illegitimate recombination took place at non-hotspot sites having only short sequences of homology (<4 bp). Thus, t he level of ligase activity affects the frequency of illegitimate recombina tion, the length of sequence homology at the recombination sites, and the p reference for recombination at hotspots, at least after W irradiation. Thes e observations support our hypothesis that the illegitimate: recombination that generates lambda bio-transducing phages is mediated bp tire DNA break- and-join mechanism.