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, W e demonstrate in this paper that Beta protein of phage A generates recombin ants 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% recombina nts among treated cells. Mechanistically, it appears that Beta protein, a R ad52-like protein, binds and anneals the ssDNA donor to a complementary sin gle-strand near the DNA replication fork to generate the recombinant, This type of homologous recombination with ssDNA provides new avenues for studyi ng and modifying genomes ranging from bacterial pathogens to eukaryotes, Be ta protein and ssDNA may prove generally applicable for repairing DNA in ma ny organisms.