Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR) at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα) but only requires a phage annealing protein (RecT or Redβ). Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein.Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ.In a search for a simple way to use homologous recombination in E. coli for DNA engineering, we found that the phage protein pairs of either RecE/RecT, from the Rac prophage, or Redα/Redβ from λ, phage, mediate very useful double stranded break repair (DSBR) in the absence of RecBC [1]. Concomitantly, Murphy [2] reported similar findings from studies with λ Red recombination. Notably, we found that this DSBR requires only very short homology regions for efficient recombination and the ratio of homologous to non-homologous recombination is very high [1]. Together with other advantages, these remarkable prope