A novel recombination pathway initiated by the Mre11/Rad50/Nbs1 complex eliminates palindromes during meiosis in Schizosaccharomyces pombe

Abstract

DNA palindromes are rare in humans but are associated with meiosis-specific translocations. The conserved Mre11/Rad50/Nbs1 (MRN) complex is likely directly involved in processing palindromes through the homologous recombination pathway of DNA repair. Using the fission yeast Schizosaccharomyces pombe as a model system, we show that a 160-bp palindrome (M-pal) is a meiotic recombination hotspot and is preferentially eliminated by gene conversion. Importantly, this hotspot depends on the MRN complex for full activity and reveals a new pathway for generating meiotic DNA double-strand breaks (DSBs), separately from the Rec12 (ortholog of Spo11) pathway. We show that MRN-dependent DSBs are formed at or near the M-pal in vivo, and in contrast to the Rec12-dependent breaks, they appear early, during premeiotic replication. Analysis of mrn mutants indicates that the early DSBs are generated by the MRN nuclease activity, demonstrating the previously hypothesized MRN-dependent breakage of hairpins during replication. Our studies provide a genetic and physical basis for frequent translocations between palindromes in human meiosis and identify a conserved meiotic process that constantly selects against palindromes in eukaryotic genomes.

Figure 1.—

Genetic markers used in this study. (A) ade6 alleles. The ade6-3034 and ade6-3036 alleles, described by Farah et al. (2002), consist of one or two copies, respectively, of an 80-bp DNA segment inserted at the BamHI site (bp 24) of the ade6 ORF (GenBank accession no. M37264). The DNA corresponds to 76 bp from the S. cerevisiae mat-a-stk locus (bp 2044–2119 of GenBank accession no. V01313; Ray et al. 1991), plus a 5′ 4-bp BamHI compatible extension. The inserted DNA is not drawn to scale. Both alleles confer an Ade⁻ phenotype. The ade6-M26 allele is a G-to-T transversion at bp 136 of the ade6 ORF and the ade6-M375 allele is a G-to-T transversion at bp 133 of the ade6 ORF (Szankasi et al. 1988). The common test allele ade6-52 is a G-to-A transition at bp 796 of the ade6 ORF (M. E. Fox, unpublished results) and is separated by 773 and 661 bp from ade6-3036 and ade6-M26, respectively. (B) Markers flanking ade6. The ura4⁺-aim insertion and tps16-23 (temperature-sensitive) markers were described previously (Grimm et al. 1994); tps16 is identical to ags1, 56.1 kb centromere distal to ade6 (Hochstenbach et al. 1998). ura4⁺-aim is an insertion of a 1.8-kb ura4⁺ cassette ∼15 kb from ade6. (0) indicates the absence of an insertion. The diagram is not drawn to scale.

Figure 2.—

Early (Rad50-dependent) and late (Rec12-dependent) DSBs at or near the M-pal. DNA was prepared at the indicated times after thermal induction (34°) of pat1-114 meiosis, digested with PacI, separated by pulsed-field gel electrophoresis, blotted, and probed from the right as indicated in H. Size markers (in kilobases) are in lane M. (A) Strain GP4653 [ade6-3036 (M-pal) rad50⁺ rec12⁺]. (B) Strain GP4207 [ade6-3034 (SI) rad50⁺ rec12⁺]. (C) Strain GP4388 (ade6-3036 rad50⁺ rec12Δ). (D) Strain GP4899 (ade6-3034 rad50⁺ rec12Δ). (E) Strain GP3279 (ade6-3036 rad50S rec12⁺). (F) Strain GP3276 (ade6-3034 rad50S rec12⁺). (G) Strain GP4802 (ade6-3036 rad50S rec12Δ). (H) Diagram of the PacI fragment containing the ade6 gene. Shown are the positions of the M-pal or SI, ade6 ORF, and probe used. Rec12-dependent meiosis-specific DSBs are indicated by solid circles. M-pal-dependent breaks are indicated by an asterisk. Nonspecific hybridization, present at each time point, is indicated by open circles. High-molecular-weight DNA, between 2 and 3 hr likely representing replicating branched DNA, is indicated by open vertical bars at the top of the scans.

Figure 3.—

Model for the meiotic elimination of the M-pal by the MRN and Rec12-dependent pathways. (A) MRN-dependent pathway. During premeiotic S phase, the M-pal extrudes on the lagging DNA strand, generating a hairpin (Leach 1994). The MRN complex recognizes and cleaves the hairpin. Cleaveage by the MRN complex could happen at the base of the stem, thus generating a homologous 3′-end ready for strand invasion. Alternatively, the MRN complex could cleave inside the hairpin, generating a heterologous 3′-end that needs to be removed by Rad16/Swi10 to allow the subsequent steps of recombination. In the presence of the rad50S allele at 25°, cleavage of the hairpin is severely impaired but subsequent processing of the DSB is active. In the presence of the rad32-D65N allele, cleavage of the hairpin is eliminated, and it is unknown whether DSB end processing is inhibited. These MRN-dependent DSBs are likely the lesions responsible for generating the Rec12-independent Ade⁺ recombinants. The MRN-dependent DSBs may also recombine with the newly synthesized sister chromatid, as indicated by the dashed arrow, and therefore may not generate an observable recombinant. (B) Rec12-dependent pathway. DNA in the vicinity of the M-pal is devoid of nucleosomes (cross-hatched circles) perhaps upon extrusion of the M-pal. This allows Rec12 to introduce DSBs in the vicinity of the hairpin after premeiotic DNA synthesis. The rad50S allele at 25° has negligible effects on this pathway as observed in crosses involving either the SI control allele or the M26 hotspot (Tables 1, 2, and 4). In the presence of the rad32-D65N allele (or the rad50S allele at 34°), processing of Rec12-generated DSBs in ade6 and elsewhere in the genome is blocked and spore formation is severely defective.