High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides

Abstract

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.

Figure 1

The galK DNA segment. At the top is a sequence from the galK gene illustrating an amber mutation galKam. The sequence shown encodes amino acids 135 through 156. The amber mutation is at tyr-145. Below is a 70-base oligo with a sequence identical, except for one base, to the non-template strand in galKam. It was used to replace the mutation with the wild-type allele.

Figure 2

The defective λ prophage. The prophage contains λ genes from cI to int (4). Some λ genes are not shown for clarity. A deletion (Δ) removes cro through attR from the right side of the prophage. p_L and the arrow indicate the early left promoter and transcript used to express Gam, Beta, and Exo. The CI857 repressor prevents p_L expression at 32^o but is inactive at 42^o. attL and attR indicate the left and right junction of the prophage. Gene replacements with the cat cassette and a simple deletion Δ(gam-N) are shown below the prophage map and are located to the left of the strain name they represent. For the experiment, cells were induced for 15 min at 42°C and electroporated with 200 ng of the 70-base oligo of Fig. 1. The Gal⁺ recombinants were selected on minimal galactose medium, and the numbers are normalized per 10⁸ cells surviving each electroporation.

Figure 3

Correlation of single strand recombination with direction of replication. The bidirectionally replicated E. coli chromosome is illustrated as a circle with the directions of replication from the origin (oriC) toward the terminus (ter) indicated by arrows outside the circle. Arrows inside the circle indicate the direction of transcription of each gene. Mutations in each of these genes were corrected by oligo recombination (see Tables 1 and 2). The relative efficiency of two complementary oligos (cw or cc) was compared for each mutation (see Table 2). The oligo generating the greater (>) recombination is indicated.