End-processing during non-homologous end-joining: a role for exonuclease 1

Abstract

Non-homologous end-joining (NHEJ) is a critical error-prone pathway of double strand break repair. We recently showed that tyrosyl DNA phosphodiesterase 1 (Tdp1) regulates the accuracy of NHEJ repair junction formation in yeast. We assessed the role of other enzymes in the accuracy of junction formation using a plasmid repair assay. We found that exonuclease 1 (Exo1) is important in assuring accurate junction formation during NHEJ. Like tdp1Δ mutants, exo1Δ yeast cells repairing plasmids with 5'-extensions can produce repair junctions with templated insertions. We also found that exo1Δ mutants have a reduced median size of deletions when joining DNA with blunt ends. Surprisingly, exo1Δ pol4Δ mutants repair blunt ends with a very low frequency of deletions. This result suggests that there are multiple pathways that process blunt ends prior to end-joining. We propose that Exo1 acts at a late stage in end-processing during NHEJ. Exo1 can reverse nucleotide additions occurring due to polymerization, and may also be important for processing ends to expose microhomologies needed for NHEJ. We propose that accurate joining is controlled at two steps, a first step that blocks modification of DNA ends, which requires Tdp1, and a second step that occurs after synapsis that requires Exo1.

Figure 1.

Plasmid repair efficiency in wild-type and exo1Δ strains. Plasmid YCplac111 was linearized with HindIII, PstI or SmaI, and transfected into BY4741 (WT) or in an exo1Δ derivative by electroporation. Repair frequencies for each genotype are expressed as the ratio of colonies obtained with linear DNA divided by colonies obtained with circular DNA ×100. The results shown are the mean of at least three independent transfections. The error bars indicate SEM.

Figure 2.

Spectra of junctions recovered from the wild-type and exo1Δ strains. The accuracy of repair of plasmids of linearized DNA with 5′-extensions (HindIII, A and EcoRI, B) or 3′-extensions (PstI, C) was determined in WT and exo1Δ deleted strains. Plasmid DNA was isolated, and the repair junction was amplified by PCR. PCR products were digested using the same restriction enzyme used to linearize the DNA, and samples that could be digested were scored as accurately repaired. Samples that failed to digest were analyzed by DNA sequencing. The enzyme recognition sites are highlighted in bold. The junctions with insertions are underlined in gray. Stars denote accurately repaired junctions.

Figure 3.

Insertions generated in exo1Δ strains occur through the NHEJ pathway and require POL4. (A) HindIII linearized YCplac111 was transfected into strains carrying a deletion of yku80Δ or of both exo1Δ and yku80Δ. As in Figure 1, transformation efficiency was normalized to undigested YCplac111. The efficiency of transformation was substantially reduced in the yku80 strain compared with the wild-type strain and the exo1Δ yku80Δ strain showed a similar reduction compared with wild-type cells. (B) Accuracy of the repair of HindIII-linearized YCplac111 DNA was assessed in plasmids recovered from yku80Δ and exo1Δ yku80Δ strains. The data from the yku80Δ strain includes earlier published samples from our laboratory, as well as 100 additional isolates (23). The deletions in the exo1Δ yku80Δ strain ranged from 1 to 23 nt in length. (C) Accuracy of the repair of HindIII-linearized YCplac111 DNA was assessed in pol4Δ and pol4Δ exo1Δ strains. The data from the pol4Δ strain includes earlier published samples from our laboratory, as well as 100 additional isolates (23).

Figure 4.

Repair of plasmid with blunt ends leads to smaller deletions in exo1Δ cells. (A) After transformation with YCplac111 DNA linearized with SmaI, plasmids were isolated from cells with the indicated genotypes. A total of 100 plasmids were analyzed from each genotype. The total numbers of accurately repaired and misrepaired plasmids are indicated. The number of misrepaired plasmids was not statistically different between any of the four genotypes examined. (B) The isolates carrying deletions of a given size is shown on the dot plot for wild-type, exo1Δ, tdp1Δ, pol4Δ and exo1Δ pol4Δ mutants. Each dot represents a single isolate with the indicated deletion size, and accurately repaired junctions are not presented. The median deletion size is shown as a solid gray line, and was calculated based on the colonies carrying deletions. Colonies with accurately repaired junctions are not included in the calculation of the median and are not shown on the dot plot.

Figure 5.

Deletion of EXO1 along with TDP1 results in an additive increase in additions. The accuracy of repair of plasmids of linearized DNA with 5′-extensions (HindIII, A and EcoRI, B) or 3′-extensions (PstI, C) was determined in exo1Δ tdp1Δ deleted strains. Results with wild-type and exo1Δ are the data presented in figure 2, and the data from the tdp1Δ deleted strain was published earlier (23). The overall level of inaccurate repair for exo1Δ tdp1Δ was compared with exo1Δ using Fisher’s exact test. For the HindIII digested DNA, P = 0.034; and for the EcoRI digested DNA, P = 0.008. The difference between any of the mutants with plasmid linearized with PstI was not significant.

Figure 6.

EXO1 is not required for tdp1Δmediated stimulation of joining blunt ends. (A) YCplac111 digested with SmaI was transfected into WT, exo1Δ, tdp1Δ or exo1Δ tdp1Δ strains. Repair frequencies were normalized with transfection of undigested YCplac111. The results shown are the mean of at least three independent transfections; error bars indicate SEM. (B) Plasmids were recovered from 100 independent colonies from wild-type, exo1Δ, tdp1Δ and exo1Δ tdp1Δ mutant strains. Data from the wild-type and single mutants were as presented in Figure 4B. As in Figure 4B, each dot represents a single isolate with the indicated deletion size, and accurately repaired junctions are not presented.

Figure 7.

A model for the roles of Exo1 in accurate joining of cohesive ends. DNA ends with 5′-extensions are potentially substrates for filling in by Pol4 or another DNA polymerase. We previously suggested that the filling reaction might be largely prevented in the presence of active Tdp1, due to the removal of a nucleoside, and the generation of a 3′-phosphate (this reaction is not shown for simplicity). If Tdp1 does not act, and a DNA polymerase fills in the extension, synapsis and isomerization can lead to a structure with a 5′-flap. The flap can be removed by either the 5′→3′-exonuclease or the flap endonuclease activity of Exo1, leading to the recovery of an error-free product. The filling-in reaction cannot be directly reversed by Exo1, since it does not have 3′→5′-exonuclease activity.