Engineering ssODN Annealing at DNA Replication Forks

Yamamoto et al., 1992 Yamamoto T.

Moerschell R.P.

Wakem L.P.

Ferguson D.

Sherman F. Parameters affecting the frequencies of transformation and co-transformation with synthetic oligonucleotides in yeast. Carr et al., 2012 Carr P.A.

Wang H.H.

Sterling B.

Isaacs F.J.

Lajoie M.J.

Xu G.

Church G.M.

Jacobson J.M. Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection. −4 CFU), and we observed ∼0.1% ade2 CFUs for msh2Δ ( We hypothesized that Rad51-dependent ssODN recombination and incorporation of ssODNs at the DNA replication fork are two distinct pathways in eukaryotes ( Figure 1 A). Sherman and colleagues observed high frequencies of co-transformation for two ssODNs targeted within a cyc1 mutant gene when one of the ssODNs included a selectable mutation (), which suggests that the two ssODNs were incorporated at the same DNA replication fork, and a finding also observed in E. coli (). To test ssODN gene editing at the replication fork, we constructed an experimental locus on chromosome 15 with a defined DNA replication direction by placing URA3 proximal to the origin of replication ARS1516 (Ori) directly adjacent to ADE2, which confers a colorimetric phenotype (wild-type [WT], white; mutant, red) ( Figure 1 C). We transformed cells with ssODNs targeting the predicted lagging strand of both URA3 and ADE2 and assayed the frequency of ade2 colony-forming units (CFUs). We did not detect ade2 CFUs for the WT strain (limit of detection [LOD] = 10CFU), and we observed ∼0.1% ade2 CFUs for msh2Δ ( Figure 1 D). After 5-FOA selection to enrich for a competent subpopulation in ura3 clones, we observed 22% and 27% of CFUs with ssODN-derived ade2 mutations for WT and msh2Δ, respectively. To determine if the ARF enhancement is due to a coupled replication fork annealing mechanism we tested a marker-target pair (RPL28-ADE2) separated on different chromosomes ( Figures S1 G and S1H). After selection for rpl28 mutants, we recovered ade2 mutants in only 0.4% of CFUs, and overexpression of Rad51 led to a ∼100% increase (0.8%). Thus, ssODN incorporation is enhanced in ura3-edited competent cells where downstream modifications are kinetically favored along a DNA replication fork in a localized chromosomal region.

−3 CFU). The effect of rad59Δ was epistatic to rad52Δ (rad52Δrad59Δ). We observed varying degrees of suppression of rad59Δ and rad52Δrad59Δ with overexpression of Rad51, Rad52, Rad59, or λ Red Beta. Since loss of Rad52 impairs Rad51-dependent HR ( Sung, 1997 Sung P. Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase. To assess the impact of HR factors on the proposed replication fork annealing mechanism, we created a set of KO and overexpression strains for HR genes and assayed ssODN incorporation at the Ori-URA3-ADE2 locus. Unlike the RPL28-ADE2 interchromosomal pair, overexpression of Rad51 decreased ARF from ∼22% to ∼5%, whereas deletion of Rad51 increased ARF to ∼30% ( Figure 1 E). Application of the Rad51 inhibitor RI-1 also enhanced ARF ( Figure S1 I). Overexpression of the SSAP Rad52 increased ARF to ∼29%, whereas deletion of Rad52 showed a neutral effect. Overexpression of the SSAP Rad59 decreased ARF to ∼16%, whereas we did not observe ade2 mutants in rad59Δ (LOD = 10CFU). The effect of rad59Δ was epistatic to rad52Δ (rad52Δrad59Δ). We observed varying degrees of suppression of rad59Δ and rad52Δrad59Δ with overexpression of Rad51, Rad52, Rad59, or λ Red Beta. Since loss of Rad52 impairs Rad51-dependent HR (), we observed a higher ARF with Rad51 overexpression in rad59Δ (4.6%) than rad52Δ (0.6%) or rad52Δrad59Δ (1.5%). Overexpression of Rad52 showed an equivalent ARF in all three strains (∼10%). The effect of Rad59 overexpression was enhanced in rad51Δ, rad52Δ, and rad52Δrad59Δ, potentially because of reduced competition from Rad51-dependent HR in these strains. Notably, λ Red Beta enhanced ARF to ∼29% in WT and recovered rad59Δ and rad52Δrad59Δ to ∼13%. In summary, our data show that ssODN annealing at the replication fork is distinct from Rad51-mediated ssODN recombination, enhanced in the rad51Δ background, and requires an SSAP (e.g., Rad59, Beta).