Mechanism and Control of Meiotic DNA Double-Strand Break Formation in S. cerevisiae

Abstract

Developmentally programmed formation of DNA double-strand breaks (DSBs) by Spo11 initiates a recombination mechanism that promotes synapsis and the subsequent segregation of homologous chromosomes during meiosis. Although DSBs are induced to high levels in meiosis, their formation and repair are tightly regulated to minimize potentially dangerous consequences for genomic integrity. In S. cerevisiae, nine proteins participate with Spo11 in DSB formation, but their molecular functions have been challenging to define. Here, we describe our current view of the mechanism of meiotic DSB formation based on recent advances in the characterization of the structure and function of DSB proteins and discuss regulatory pathways in the light of recent models.

Keywords: DNA recombination; Spo11; double-strand break; meiosis; phase separation.

FIGURE 1

Overview of meiosis and meiotic recombination. (A) Schematic of the formation of haploid gametes from a diploid cell with a single pair of homologous chromosomes. DSB formation and recombination promote homolog pairing and lead to the exchange of chromosomal fragments (crossovers) in the context of synapsed chromosomes. (B) Meiotic recombination is initiated by Spo11-mediated DSB formation and leads to the formation of crossovers via a ZMM-dependent double Holliday Junction (dHJ) resolution pathway or non-crossovers by synthesis-dependent strand annealing. (C) Relationships between meiotic recombination and higher-order chromosome structure. DSB formation happens in the context of the loop-axis structure. As recombination progresses, the SC polymerizes between the axes and is disassembled prior to chromosome segregation. Axis proteins Red1 (red ovals) and Hop1 (yellow ovals) are shown. (D) In metaphase I, homologs are held together through chiasmata and sister chromatid cohesion.

FIGURE 2

DSB formation in S. cerevisiae. (A) The distribution of meiotic DSBs is influenced by a combination of factors that operates at various size scales (Pan et al., 2011). Spo11 footprint indicates the expected occupancy of Spo11 on DNA based on structural modeling. (B) The tethered loop-axis model for DSB formation. Spp1 binds to H3K4me2/3 enriched around DSB hotspots and connects it to the chromosome axis through interaction with Mer2. Axis proteins Red1 (red ovals) and Hop1 (yellow ovals) are shown. (C) Ten DSB proteins in S. cerevisiae.

FIGURE 3

Mechanism of Topo VI. (A) Chemistry of strand cleavage and re-sealing in Topo VI. (B) Cartoon of the Topo VI heterotetramer. (C) Domain structure of the A and B subunits of Topo VI. (D) Structure of Topo VI (PDB: 2Q2E) showing the expected position of the G-segment within the groove formed by the A subunits (Corbett et al., 2007). (E) Catalytic cycle of Topo VI through a two-gate mechanism. ATP-dependent dimerization of the GHKL domain upon sequential or simultaneous binding to gate (G) and transfer (T) DNA duplexes is communicated to the A subunit to activate DSB formation. Topo VI can undergo multiple catalytic cycles without dissociation from the G-segment.

FIGURE 4

The Spo11 core complex. (A) Cartoon illustrating the arrangement of the different subunits in the core complex. (B) Domain structure of Rec104, Rec102, Spo11, and Ski8. The red dotted lines connecting two proteins represent their respective interaction domains. The region of Rec104 that interacts with Rec102 is predicted based on crosslinking-mass spectrometry, other interaction regions were validated by mutagenesis (Arora et al., 2004; Cheng et al., 2009; Claeys Bouuaert et al., 2021). The position in Spo11 of the catalytic tyrosine Y135 and metal-ion coordinating residue E233 are shown. (C) Proposed dynamics of the interaction between the core complex and DNA based on in vitro binding activities and analogy with Topo VI (Claeys Bouuaert et al., 2021). After DSB formation, Spo11 remains bound to the DSB through covalent and non-covalent interactions. (D) Inverted repeat sequences form cruciforms that fold into three-dimensional structures that are similar to two overlapping DNA duplexes (PDB: 1DCW) (Eichman et al., 2000).

FIGURE 5

The MRX complex. (A) Domain structure of Mre11, Rad50, and Xrs2, and their protein-protein interacting regions (red dotted lines). (B) Cartoon illustrating the structural arrangement of the MRX complex and the conformational dynamics upon ATP hydrolysis. In an ATP-bound state, the nuclease domain of Mre11 does not access DNA. However, after ATP hydrolysis by Rad50, a conformational change exposes the nuclease domain of Mre11 to DNA. Sae2 is illustrated here as interacting with Rad50 based on Cannavo et al. (2018), but interactions with Xrs2 have also been demonstrated (Liang et al., 2015). (C) Model for DSB resection by MRX. Endonuclease activity of Mre11 directed on the 5′-strand is followed by bi-directional resection through the 3′-5′ exonuclease activity of Mre11 and the 5′-3′ exonuclease activity of ExoI or Dna2-Sgs1 in vegetative conditions or ExoI in meiosis.

FIGURE 6

The RMM proteins. (A) Domain structure of Rec114, Mei4, and Mer2 with regions involved in protein-protein and protein-DNA interactions (Claeys Bouuaert et al., 2021). Numbered blocks indicate conserved sequence motifs (Kumar et al., 2010; Tessé et al., 2017). (B) Schematic of the Rec114—Mei4 complex. (C) Structure of the Pleckstrin-homology domain of mouse REC114 (PDB: 6HFG) (Kumar et al., 2018). Residues in gray are the conserved motifs highlighted in (A).

FIGURE 7

Structural components of the meiotic chromosome axis. (A) Domain structure of Hop1 and Red1. The C-terminal-domain (CTD) of Hop1 contains a closure motif. (B) Hop1 forms an oligomer through intermolecular interactions between the HORMA domain and the closure motif (West et al., 2018). The Red1 coiled-coil domain forms a parallel-antiparallel tetramer that can form a filament structure by end-to-end polymerization (West et al., 2019).

FIGURE 8

Model for the assembly of the meiotic DSB machinery. (A) DNA-dependent condensation of Rec114—Mei4 and Mer2 leads to the formation of large mixed nucleoprotein structures along the chromosome axis. These condensates act as a platform to recruit the Spo11 core complex, MRX, and perhaps other regulatory proteins (Claeys Bouuaert et al., 2021). This model explains the observation that Spo11 often makes closely spaced double DSBs separated with a 10-bp periodicity (Johnson et al., 2021). (B) Condensate-embedded core complexes may assist DNA repair by holding broken ends in the vicinity of one another. The condensates could also hold the broken chromatids through association with the base of the loops, independently of whether the DNA ends themselves are embedded. Axis proteins Red1 (red ovals) and Hop1 (yellow ovals) are shown.

FIGURE 9

Overlapping regulatory circuits control DSB formation. (A) (1) DSB formation is tied to cell cycle control through dependence on CDK and DDK phosphorylation of Mer2. (2) Replication stress inhibits DSB formation by different mechanisms through activation of the Mec1 checkpoint kinase. (3) Replication also positively impacts DSB formation by promoting Mer2 phosphorylation. (4) Recombination defects activate Mec1, which extends prophase by preventing Ndt80 activation, thereby producing a positive feedback loop. (5) Activation of the DNA-damage response kinase Tel1 inhibits further DSB formation, thereby creating a negative feedback loop. (6) Hotspot competition (Tel1-independent) and DSB interference (Tel1-dependent) impact spatial distribution of DSB formation, which limits the coincident formation of two DSBs in cis within a 100-kb range or in trans between allelic regions of sister chromatids or homologs. (7) Homolog engagement shuts down DSB formation through SC-dependent removal of DSB proteins. (8) Exit of pachytene following Ndt80 activation ends the DSB-permissive period. (B) Positive and negative impacts of DNA replication on DSB formation. DDK is bound to the replisome via interactions with the fork protection complex (FPC). Phosphorylation of Mer2 in regions that have undergone replication promotes the assembly of the DSB machinery and DSB formation (Murakami and Keeney, 2014). However, replication stress activates Mec1 and inhibits DSB formation by reducing Spo11 transcription, inhibiting DDK via Rad53, and independently inhibiting chromatin association of several DSB proteins (Blitzblau and Hochwagen, 2013).

FIGURE 10

The condensate model for hotspot competition, DSB interference, and homolog engagement. (A) The model suggests that hotspot competition is mediated prior to DSB formation through partitioning of RMM proteins into condensates, locally depleting pools of free DSB proteins. (B) DSB formation activates Tel1, which inhibits local DSB formation. (C) SC assembly leads to the removal of Hop1 and DSB proteins from the axis, thereby shutting down further DSB formation.