Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells.
BACKGROUND
The phage protein pairs, RecE/RecT from Rac or Redalpha/Redbeta from lambda, initiate efficient double strand break repair (DSBR) in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined.


RESULTS
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 Redalpha) but only requires a phage annealing protein (RecT or Redbeta). 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.


CONCLUSIONS
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 Redalpha/Redbeta.
Zhang
Youming
Y
Gene Expression Program, EMBL, Meyerhofstr, 1, 69117, Heidelberg. youming.zhang@genebridges.com <youming.zhang@genebridges.com>
Muyrers
Joep P P
JP
Rientjes
Jeanette
J
Stewart
A Francis
AF
D000818:Animals; D001435:Bacteriophages / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000737:chemistry; D011995:Recombination, Genetic; D013234:Stem Cells / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D014764:Viral Proteins