High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides.
Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology. We demonstrate in this paper that Beta protein of phage lambda generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant. This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms.
Ellis
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HM
Gene Regulation and Chromosome Biology Laboratory, Division of Basic Sciences, National Cancer Institute at Frederick, P.O. Box B, Frederick, MD 21702, USA.
Yu
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DiTizio
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Court
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D000595:Amino Acid Sequence; D001483:Base Sequence; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011938:Rec A Recombinases / Q000032:analysis; D011995:Recombination, Genetic; D014764:Viral Proteins
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C118730:beta protein, Bacteriophage lambda; D011938:Rec A Recombinases