Yeast Tdp1 regulates the fidelity of nonhomologous end joining

Abstract

Tyrosyl-DNA-phosphodiesterase 1 (Tdp1) can disjoin peptides covalently bound to DNA. We assessed the role of Tdp1 in nonhomologous end joining (NHEJ) and found that linear DNA molecules with 5' extensions showed a high frequency of misrepair in Deltatdp1 cells. The joining errors in Deltatdp1 cells were predominantly 2-4 nucleotide insertions. Ends with 3' extensions or blunt ends did not show enhanced frequencies of errors, although Deltatdp1 cells repaired blunt DNA ends with greater efficiency than WT cells. We found that insertions required Ku80 and DNA ligase IV, as well as polymerase IV. Our results show that yeast Tdp1 is a component of the NHEJ pathway. We suggest that Tdp1p 3' nucleosidase activity regulates the processing of DNA ends by generating a 3' phosphate, thereby restricting the ability of polymerases and other enzymes from acting at DNA ends. In support of this model, we found that overexpression of Tpp1, a yeast DNA 3' phosphatase, also leads to a higher frequency of insertions, suggesting that the generation of a 3' phosphate is a key step in Tdp1-mediated error prevention during NHEJ.

Fig. 1.

NHEJ efficiency and repair fidelity in WT and tdp1 deleted strains. (A) Plasmid YCplac111 linearized with HindIII, EcoR1, or Pst1 were transfected into BY4741 (WT) or a Δtdp1 derivative by using electroporation. Repair frequencies for each genotype are expressed as the ratio of colonies obtained with linear DNA divided by colonies obtained with uncut DNA × 100. The results shown are the mean of at least three independent transfections performed with the same reagents; error bars indicate SEM. (B) Accuracy of repair of linear DNA with 5′ extensions (HindIII and EcoRI) or 3′ extensions (PstI) was determined in WT and Δtdp1 strains. After transfection with YCplac111-linearized DNA, plasmids were isolated from colonies as described in Methods. Joints were amplified by PCR and redigested by the same restriction enzyme used to linearize the DNA. PCR samples that redigested were deemed accurately repaired. Samples that failed to digest were analyzed by DNA sequencing. The experiments analyzed 100 colonies per condition by design. Accurately repaired junctions are indicated in the line marked with a star, with spaces to allow the alignment of insertions. Junction sequences in red indicate insertions based on the left side filling in, and sequences in blue are insertions based on right-side fill-in reactions, although some assignments are arbitrary. A single nontemplated insertion of a cytosine was recovered in the Δtdp1 strain when transfected with HindIII-linearized DNA (indicated by the underlined C). The number of identical sequences recovered is indicated for each sequence.

Fig. 2.

Effects of alterations in DNA repair pathways on inaccurate end joining when TDP1 is deleted. (A) Derivatives of BY4741 carrying deletions in NHEJ functions and/or TDP1 were constructed, and the efficiency of repair of HindIII-linearized YCplac111 was assessed. As in Fig. 1A, repair frequencies for each genotype are expressed as the ratio of colonies obtained with linear DNA divided by colonies obtained with uncut DNA × 100. Note that the transformation efficiency is shown on a logarithmic scale. (B) Accuracy of the repair of HindIII-linearized YCplac111 DNA was assessed in Δyku80 and Δyku80 Δtdp1 strains. As indicated, no misrepaired plasmids were recovered. Similar results were obtained with Δdnl4 and Δdnl4 Δtdp1 strains (Fig. S2). Accurately repaired junctions are indicated in the line marked with a star. (C) Accuracy of the repair of HindIII-linearized YCplac111 DNA was assessed in Δpol4 and Δpol4 Δtdp1 strains. (D) Accuracy of the repair of HindIII-linearized YCplac111 DNA was assessed in Δrad52 and Δrad52 Δtdp1 strains. (E) A derivative of JN362a was constructed carrying either a URA3 disruption of Δtdp1 or a replacement of the WT TDP1 gene with an allele encoding a His182Ala missense mutation. A more complete description of the junction sequences obtained is presented in Fig. S4. (F) TPP1 overexpression leads to misrepair of linearized plasmids in WT cells. By4741 cells carrying either pTW375 (WT yeast TPP1 under the control of the yeast Adh1 promoter) (24) or pTW375^D35A (as pTW375, but with a Tpp1 mutation changing Asp 35 to Ala) (24) were transfected with YCplac111 that had been linearized with HindIII. Accuracy of repair of the linearized DNA was determined as described in the legend for Fig. 1. The overall repair efficiency was similar to results presented in Fig. 1 for WT cells.

Fig. 3.

Analysis of plasmids obtained following transformation with DNA with incompatible ends. (A) Plasmid yCPlac11 was cut with HindIII and Sal1 and introduced into WT and Δtdp1 strains, which were analyzed by DNA sequencing for the nature of the repaired junctions. The events were rationalized by assuming fill-in reactions of the HindIII end or the SalI end of the molecule. Filling in of the HindIII end is shown as red nucleotides, and filling in of the SalI end is shown with blue nucleotides. For WT cells, 27/50 showed additions consistent with partial filling in of one of the two sides and 1/50 showed a complete filling in of the extensions By contrast, 33/50 of the plasmids from tdp1 showed complete filling in, and 11 more showed almost complete filling in. No deletions were recovered from tdp1 cells. A complete sequence analysis is presented in Fig. S6a. (B) Colonies obtained from transformation with yCPlac11 digested with HindIII and Pst1 were analyzed for the nature of the repaired junctions. Events on the HindIII cut end were rationalized as before by assuming filling in of the 5′ extension (shown as red nucleotides), whereas events at the Pst1 side were rationalized on the basis of preserving the 3′ extension (shown with blue nucleotides). Seven large deletions (> 10 nucleotides) were recovered in WT cells compared to none in tdp1 cells. A complete sequence analysis is presented in Fig. S6b.

Fig. 4.

NHEJ efficiency and repair fidelity in WT and tdp1-deleted strains transfected with DNA with blunt ends. (A) YCplac111 cut with Sma1 was transfected into yeast by using the same methods as in Figs. 1 and 2. (B) Strains transfected with DNA with blunt ends show considerably less accurate repair than DNA with cohesive ends. Accurately repaired junctions are indicated in the line marked with a star. For WT cells 61/100 were repaired accurately, whereas 67/100 were repaired accurately in Δtdp1 strains, an insignificant difference (p = 0.462 by using Fisher’s exact test). There was no obvious preference for the size of the deletions in WT versus Δtdp1 strains. (C) YCplac111 was cut with both HindIII and SalI. All samples were analyzed by DNA sequencing, with 50 independent plasmids each from WT and Δtdp1 strains analyzed. The figure shows a summary of the insertion and deletion events; a more complete listing of the recovered events is shown in Fig. S3. The overall frequency of repaired plasmids with insertions was statistically different between WT and Δtdp1 strains (all Δtdp1 strains carried insertions, p < 0.0001) as was the frequency of deletions in WT cells.

Fig. 5.

A model for suppression of additions by Tdp1 nucleosidase activity. We suggest that Tdp1 acts directly on 3′ DNA ends during NHEJ. The nucleosidase activity of Tdp1 removes a single nucleoside, leaving a terminal 3′ phosphate. This blocks polymerization and ligation until the phosphate is removed. Complete repair of the double-strand break will require removal of the phosphate by Tpp1 or removal of the nucleotide phosphate by a nuclease activity such as Apn1. Polymerization is required to repair the gap generated by Tdp1 nucleosidase activity prior to ligation. Recent results suggest that polymerase activity is required for efficient sealing of cohesive ends with 5′ extensions (33), supporting the hypothesis that some nucleotides are removed during the repair of DNA with cohesive ends. When Tdp1 activity is absent, DNA polymerases can extend a primed template, leading to nucleotide insertions. The simplest hypothesis based on our results is that the insertions result from additions by Pol4p.