Nonhomologous End-Joining with Minimal Sequence Loss Is Promoted by the Mre11-Rad50-Nbs1-Ctp1 Complex in Schizosaccharomyces pombe

Abstract

While the Mre11-Rad50-Nbs1 (MRN) complex has known roles in repair processes like homologous recombination and microhomology-mediated end-joining, its role in nonhomologous end-joining (NHEJ) is unclear as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mammals have different requirements for repairing cut DNA ends. Most double-strand breaks (DSBs) require nucleolytic processing prior to DNA ligation. Therefore, we studied repair using the Hermes transposon, whose excision leaves a DSB capped by hairpin ends similar to structures generated by palindromes and trinucleotide repeats. We generated single Hermes insertions using a novel S. pombe transient transfection system, and used Hermes excision to show a requirement for MRN in the NHEJ of nonligatable ends. NHEJ repair was indicated by the >1000-fold decrease in excision in cells lacking Ku or DNA ligase 4. Most repaired excision sites had <5 bp of sequence loss or mutation, characteristic for NHEJ and similar excision events in metazoans, and in contrast to the more extensive loss seen in S. cerevisiaeS. pombe NHEJ was reduced >1000-fold in cells lacking each MRN subunit, and loss of MRN-associated Ctp1 caused a 30-fold reduction. An Mre11 dimer is thought to hold DNA ends together for repair, and Mre11 dimerization domain mutations reduced repair 300-fold. In contrast, a mre11 mutant defective in endonucleolytic activity, the same mutant lacking Ctp1, or the triple mutant also lacking the putative hairpin nuclease Pso2 showed wild-type levels of repair. Thus, MRN may act to recruit the hairpin opening activity that allows subsequent repair.

Keywords: Hermes; MRN; MRX; Mre11-Rad50-Nbs1; Mre11-Rad50-Xrs2; NHEJ; hAT; hairpin; nonhomologous end-joining; transposon.

Figure 1

A transient transfection assay to generate mutants with single-transposon insertions. (A) Cells grown under selection for the pHL2578u plasmid expressing the Hermes transposase from the nmt1 promoter under inducing conditions. The pHL2577 transposon donor plasmid contains the ends of the Hermes transposon flanking a kanamycin selectable marker (KanMX) that confers resistance to G418. Introducing the transposon plasmid into cells expressing the transposase allows insertion of the transposon into the genome, generating a target site duplication of 8 bp (indicated by thin arrows). (B) Cells expressing transposase were transformed with different amounts of the transposon plasmid. To monitor whether continuous expression of transposase impacted transposition, half of the transformed cells were plated onto either noninducing (YES) or inducing (EMM) nonselective media. The EMM plates contained supplements to complement cellular auxotrophies (Moreno et al. 1991). After 24 hr of growth to allow plasmid loss, the lawn of cells was replica plated onto medium that selects for the transposon (G418-resistance) and against the ura4⁺ and URA3⁺ genes (FOA) on the two plasmids. The yield of colonies bearing transposon insertions is shown in Table 1. aluh, adenine, leucine, uracil, and histidine; FOA, 5’-fluoroorotic acid; YES, yeast extract sucrose.

Figure 2

Removal of a Hermes transposon requires end-joining repair. (A) Transposon excision produces hairpin-capped DSB ends that must be repaired, or cells will lose essential genetic information during mitosis and die. Repair by end-joining (NHEJ or MMEJ, left panel) involves nuclease activities and ligation of the broken ends, which can be identified by PCR. Rare events can be detected by a second PCR with a set of nested primers. Repair by HR in G2 cells that retain an unexcised transposon (right panel) will regenerate the Hermes insertion. This product will not be detected in this assay due to the use of short extension times that do not allow amplification of the Hermes insertion. The arrows indicate primers for the PCR reactions. (B) KRP 3-4 cells containing a single-transposon insertion (Figure S1 in File S1) were transformed with the transposase expression plasmid to generate colonies from single cells. The individual colonies were grown to 5 × 10⁷ cells under conditions that induced transposase expression, and DNA was prepared and used in two rounds of PCR. Excision frequencies were determined by comparison to the standard curve shown in the next panel. The lanes labeled “C” are loaded with various amounts of a marker that show the size of the expected fragment. (C) A reconstruction test was performed to estimate the frequency of Hermes excision. The indicated number of WT, which lack a Hermes insertion, were mixed with 5 × 10⁷ KRP 3-4 cells bearing a transposon and used to prepare DNA. PCR to detect only the excision products (as in A) was performed in triplicate for each sample. Quantitation and comparison of the first and second PCR products indicated that excision frequencies differing by 10-fold could be distinguished over the range of 10⁻⁴ to 10⁻⁶ per cell. DSB, double-strand breaks; HR, homologous recombination; MMEJ, microhomology-mediated end-joining; NHEJ, nonhomologous end-joining; wild-type, WT.

Figure 3

Hermes excision footprint sequences indicate repair by NHEJ. Hermes excision events were monitored by cloning and sequencing the first-round PCR products from individual colonies in which transposon excision was induced. Cells bearing one of two independent transposon insertions were analyzed: KRP 3-4 cells (A) and KRP 3-3 cells (B). The PCR products for the KRP 3-4 cells are those shown in Figure 2B. The underlined sequence is the 8-bp duplication generated during transposon insertion. Base changes are indicated in red, and deletions are indicated by a colon. Most events show small deletions (0–5 nucleotides) and mutations that implicate NHEJ (Daley et al. 2005; McVey and Lee 2008). A bracket indicates the sequences used to model the mechanism of repair (Figure S4 in File S1). NHEJ, nonhomologous end-joining.

Figure 4

Efficient NHEJ requires Ku70, DNA ligase 4, and MRN. KRP 3-4 cells bearing no mutation (WT) or the indicated mutations were tested in the excision assay. Ten individual colonies for each strain were tested by two rounds of PCR. Lane “C” contains various amounts of a PCR product from wild-type DNA loaded as a size marker for the expected NHEJ fragment. The WT gels are the same as those shown in Figure 2B. The median excision frequencies and footprint sequences are shown in Figure 5 and Figure S5 in File S1, respectively. MRN, Mre11-Rad50-Nbs1; NHEJ, nonhomologous end-joining; wild-type, WT.

Figure 5

Hermes excision frequencies of different NHEJ mutants. Each data point represents the excision frequency of a single colony from the WT or mutant cells analyzed by PCR (Figure 4). The short red bars indicate the median excision frequency for the strain, which is obtained by ranking the frequencies from highest to lowest and averaging the fifth and sixth values. The differences between the WT and all strains were statistically significant (P < 0.001 for all strains by Mann–Whitney two-tailed rank sum test except for WT vs. ctp1∆, which was P = 0.0021). NHEJ, nonhomologous end-joining; wild-type, WT.

Figure 6

Mutations in the Mre11 dimerization domain, but not the nuclease domain, reduce NHEJ. Strains bearing the Hermes insertion and the indicated mre11 mutations were tested in the excision assay as in Figure 2 and Figure 4. The indicated median excision frequencies and excision footprint sequences are shown in Figure 7 and Figure S7 in File S1, respectively. NHEJ, nonhomologous end-joining; wild-type, WT.

Figure 7

Hermes excision frequencies of different mre11 mutants. Each data point represents the excision frequency from a WT or mutant cell culture analyzed by PCR (Figure 6). The red bar shows the median frequency. The differences between mre11⁺ WT and mre11-L77K and mre11-L77KL174D were statistically significant (P < 0.001 and 0.002, respectively, by Mann–Whitney two-tailed rank sum test), the difference between mre11⁺ WT and mre11-H68S was borderline for significance in this nonparametric test (P = 0.023), while the differences between mre11⁺ and mre11-H134S or ctp1∆ mre11-H134S, and between mre11-H134S and ctp1∆ mre11-H134S, were not distinguishable (P = 0.218, 0.684, and 0.143, respectively). NHEJ, nonhomologous end-joining; wild-type, WT.

Figure 8

The S. pombe homolog of in vitro hairpin nuclease Pso2 is not required for excision. S. cerevisiae Pso2 can cleave DNA hairpins in vitro (Tiefenbach and Junop 2012), suggesting that the S. pombe homolog may account for the excision healing activity in mre11 mutants that lack nuclease activity. Hermes excision was analyzed with a modified assay (see Materials and Methods) that analyzed 11 colonies. (A) A standard curve constructed using a culture of 2 × 10⁷ cells that also contained the indicated number of wild-type cells to model the excision event as in Figure 2. The lane numbers indicate genomic DNA samples that were amplified in parallel with the colonies in (B) and (C). (B) Hermes excision frequencies in the epitope-tagged mre11-13myc strains are not affected by loss of Pso2 (P = 0.84 by Mann–Whitney test). “Stds” refers to samples from the standard curve in (A) that were amplified at the same time as each of the colonies shown. The numbers above the Stds lanes refer to the lane numbers shown in (A). (C) Excision frequencies in the epitope-tagged mre11-H134S-13myc ctp1∆ double mutant strains are not affected by loss of Pso2 (P = 0.69 by Mann–Whitney test). (D) Graphical representation of the excision frequencies in (B), where the red bar denotes the median excision frequency. (E) Graphical representation of the excision frequencies in (C). Lane No., lane number; wild-type, WT.

Figure 9

Model for the role of MRN-Ctp1 in NHEJ repair of Hermes excision sites. The initial hairpin-capped ends are bound by Ku, which has end protection functions that may prevent further processing (Ramsden and Gellert 1998; Smider et al. 1998; Arosio et al. 2002). MRN-Ctp1 is proposed to bind and replace Ku and allow synapsis to bridge the two DNA ends [as in Langerak et al. (2011) and Limbo et al. (2011)] and to recruit factors that open the hairpin. Subsequent base pairing at microhomologies (yellow box) may be potentiated by MRN or other factors prior to ligation. While MRN has a known role in strand resection (McVey and Lee 2008; Nicolette et al. 2010; Paull 2010), the most frequently observed end-joining events (Figure 3) indicated that little or no strand resection occurred to expose homologies. MRN, Mre11-Rad50-Nbs1; NHEJ, nonhomologous end-joining; wild-type, WT.