PRDM9 has recently been identified as a likely trans-regulator of meiotic recombination hot spots in humans and mice1-3. The protein contains a zinc finger array that in humans can recognise a short sequence motif associated with hot spots4, with binding to this motif possibly triggering hot-spot activity via chromatin remodelling5. We now show that variation in the zinc finger array in humans has a profound effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Very subtle changes within the array can create hot-spot non-activating and enhancing alleles, and even trigger the appearance of a new hot spot. PRDM9 thus appears to be the preeminent global regulator of hot spots in humans. Variation at this locus also influences aspects of genome instability, specifically a megabase-scale rearrangement underlying two genomic disorders6 as well as minisatellite instability7, implicating PRDM9 as a risk factor for some pathological genome rearrangements.

Holliday junctions (HJs) are four-way DNA intermediates that form during homologous recombination, and their efficient resolution is essential for chromosome segregation. Here, we show that three structure-selective endonucleases, namely SLX1-SLX4, MUS81-EME1, and GEN1, define two pathways of HJ resolution in human cells. One pathway is mediated by GEN1, whereas SLX1-SLX4 and MUS81-EME1 provide a second and genetically distinct pathway (SLX-MUS). Cells depleted for SLX-MUS or GEN1 pathway proteins exhibit severe defects in chromosome segregation and reduced survival. In response to CDK-mediated phosphorylation, SLX1-SLX4 and MUS81-EME1 associate at the G2/M transition to form a stable SLX-MUS holoenzyme, which can be reconstituted in vitro. Biochemical studies show that SLX-MUS is a HJ resolvase that coordinates the active sites of two distinct endonucleases during HJ resolution. This cleavage reaction is more efficient and orchestrated than that mediated by SLX1-SLX4 alone, which exhibits a potent nickase activity that acts promiscuously upon DNA secondary structures.

PRDM9 is a major specifier of human meiotic recombination hotspots, probably via binding of its zinc-finger repeat array to a DNA sequence motif associated with hotspots. However, our view of PRDM9 regulation, in terms of motifs defined and hotspots studied, has a strong bias toward the PRDM9 A variant particularly common in Europeans. We show that population diversity can reveal a second class of hotspots specifically activated by PRDM9 variants common in Africans but rare in Europeans. These Africanenhanced hotspots nevertheless share very similar properties with their counterparts activated by the A variant. The specificity of hotspot activation is such that individuals with differing PRDM9 genotypes, even within the same population, can use substantially if not completely different sets of hotspots. Each African-enhanced hotspot is activated by a distinct spectrum of PRDM9 variants, despite the fact that all are predicted to bind the same sequence motif. This differential activation points to complex interactions between the zinc-finger array and hotspots and identifies features of the array that might be important in controlling hotspot activity.eiotic recombination is fundamentally important in ensuring correct chromosome disjunction at meiosis and in reshuffling haplotypes between generations, substantially increasing haplotype diversity within a population. Most recombination events in the human genome are clustered into narrow hotspots that can be identified indirectly from patterns of linkage disequilibrium (LD hotspots) (1), or directly through high-resolution linkage analysis in pedigrees (2) or by sperm typing (3). Genomewide comparison of LD hotspots has identified a sequence motif CCNCCNTNNCCNC associated with 40% of these hotspots; this motif appears to influence the initiation of meiotic recombination, because SNPs that disrupt the motif can down-regulate recombination (4).Recently, the meiosis-specific protein PRDM9 has been identified as a major specifier of hotspots in the human and mouse genome (5-7). PRDM9 contains a SET domain that might be responsible for activating hotspots by chromatin remodelling (8), plus a C-terminal tandem-repeat zinc-finger (ZnF) array encoded by a variable minisatellite. Evidence that PRDM9 regulates hotspots comes from the finding that the common European variant A has a ZnF array that binds, at least in vitro, to the 13-mer hotspot motif shared by many LD hotspots identified in Europeans (5, 6). Furthermore, association analyses in Hutterites (5) and Icelanders (2) have shown that individuals with variant non-A PRDM9 alleles can show genome-wide shifts in hotspot usage. These shifts suggest that ZnF variants that should not bind the PRDM9 A motif might trigger the appearance of new sets of hotspots (5), although it is possible that some of these shifts reflect additional hotspot-specification systems that only become manifest as the dosage of the PRDM9 A variant is reduced. The Icelandic study highlighted the PRDM9 C variant and its associated predicted motif CC...

The resolution of recombination intermediates containing Holliday junctions (HJs) is critical for genome maintenance and proper chromosome segregation. Three pathways for HJ processing exist in human cells and involve the following enzymes/complexes: BLM-TopoIIIa-RMI1-RMI2 (BTR complex), SLX1-SLX4-MUS81-EME1 (SLX-MUS complex), and GEN1. Cycling cells preferentially use the BTR complex for the removal of double HJs in S phase, with SLX-MUS and GEN1 acting at temporally distinct phases of the cell cycle. Cells lacking SLX-MUS and GEN1 exhibit chromosome missegregation, micronucleus formation, and elevated levels of 53BP1-positive G1 nuclear bodies, suggesting that defects in chromosome segregation lead to the transmission of extensive DNA damage to daughter cells. In addition, however, we found that the effects of SLX4, MUS81, and GEN1 depletion extend beyond mitosis, since genome instability is observed throughout all phases of the cell cycle. This is exemplified in the form of impaired replication fork movement and S-phase progression, endogenous checkpoint activation, chromosome segmentation, and multinucleation. In contrast to SLX4, SLX1, the nuclease subunit of the SLX1-SLX4 structure-selective nuclease, plays no role in the replication-related phenotypes associated with SLX4/MUS81 and GEN1 depletion. These observations demonstrate that the SLX1-SLX4 nuclease and the SLX4 scaffold play divergent roles in the maintenance of genome integrity in human cells.

DNA strand break repair by homologous recombination leads to the formation of intermediates in which sister chromatids are covalently linked. The efficient processing of these joint molecules, which often contain four-way structures known as Holliday junctions, is necessary for efficient chromosome segregation during mitotic division. Because persistent chromosome bridges pose a threat to genome stability, cells ensure the complete elimination of joint molecules through three independent pathways. These involve (1) BLM-Topoisomerase IIIα-RMI1-RMI2 (BTR complex), (2) SLX1-SLX4-MUS81-EME1 (SLX-MUS complex), and (3) GEN1. The BTR pathway promotes the dissolution of double Holliday junctions, which avoids the formation of crossover products, prevents sister chromatid exchanges, and limits the potential for loss of heterozygosity. In contrast to BTR, the other two pathways resolve Holliday junctions by nucleolytic cleavage to yield crossover and non-crossover products. To avoid competition with BTR, the resolution pathways are restrained until the late stages of the cell cycle. The temporal regulation of the dissolution/resolution pathways is therefore critical for crossover avoidance while also ensuring that all covalent links between chromosomes are resolved before chromosome segregation.