Numerical constraints and feedback control of double-strand breaks in mouse meiosis

During meiosis, the specialized cell division that generates haploid gametes for sexual reproduction, homologous chromosomes engage each other at multiple positions along their lengths via recombination initiated by DNA double-strand breaks (DSBs) (Hunter 2007). Recombination promotes homolog pairing and accurate chromosome segregation, but DSBs are potentially lethal genomic insults if repaired incorrectly or not at all. Thus, a fundamental question in chromosome biology is how meiotic cells control DSB formation to foster its essential functions but minimize untoward effects.

The simplified sequence of meiotic chromosome dynamics in many organisms can be summarized as follows: Meiotic DSBs catalyzed by SPO11 protein are processed into 3′ ssDNA tails, which are required for homology search, leading to stable interhomolog interactions and, subsequently, the formation of the synaptonemal complex (SC), a zipper-like proteinaceous structure between homologous chromosomes (e.g., see Zickler 1977; Rasmussen and Holm 1978; Maguire 1984; Zickler and Kleckner 1999; Storlazzi et al. 2010). Multiple interstitial recombination events are needed for progressive stabilization of homologous interactions and synapsis (e.g., see Smithies and Powers 1986; Weiner and Kleckner 1994; Kleckner 1995; Peoples-Holst and Burgess 2005), as inferred primarily from budding yeast, filamentous fungi, and plants (Albini and Jones 1984; von Wettstein et al. 1984; Tesse et al. 2003; Peoples-Holst and Burgess 2005). In mice, homolog synapsis is recombination-dependent; in the absence of DSBs or when certain recombination proteins are missing, SC formation is reduced, and SC that does form is frequently between nonhomologous chromosomes (Pittman et al. 1998; de Vries et al. 1999; Baudat et al. 2000; Romanienko and Camerini-Otero 2000; Petukhova et al. 2003).

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Although DSB numbers vary significantly from cell to cell (Chen et al. 2008; Cole et al. 2012), much greater intrinsic difference is seen when comparing different organisms, implying the existence of species-specific set points for DSB numbers. Species with recombination-independent pairing (Drosophila and nematodes) form relatively few DSBs (Jang et al. 2003; Mets and Meyer 2009; Rosu et al. 2011). In contrast, fungi, plants, and mammals, where recombination is required for stable pairing, make many more DSBs (e.g., Terasawa et al. 1995; Plug et al. 1996; Barlow et al. 1997; Lenzi et al. 2005; Sanchez-Moran et al. 2007; Mancera et al. 2008; Roig et al. 2010; Storlazzi et al. 2010). For example, mouse spermatocytes are estimated to make ∼200-250 DSBs on average, based on numbers of chromosome-associated foci of the strand exchange proteins RAD51 and DMC1. Of this number, only approximately one-tenth is matured into crossovers; analysis of individual recombination hot spots suggests that a large fraction of the remaining DSBs mature into noncrossovers (Cole et al. 2010; F Baudat and B de Massy, unpubl.; E de Boer, M Jasin and S Keeney, unpubl.), although the possibility of substantial sister chromatid recombination, which would not contribute to homolog pairing, has not been ruled out. The correlation of global DSB numbers with recombination dependence of pairing suggests that homolog pairing—instead of simply the requirement for crossovers—imposes a critical constraint on the minimum DSB number needed for successful meiosis.

Empirical support for this hypothesis comes from budding yeast and Sordaria macrospora, in which reduced DSB levels cause pairing and/or synapsis defects (Davis et al. 2001; Tesse et al. 2003; Henderson and Keeney 2004). However, the substantial differences in genome size and complexity between these fungi and mammals make it difficult to extrapolate from one organism to another. Within a given species, chromosome sizes vary greatly (in humans, for example, the smallest chromosome is approximately one-fifth the size of the largest chromosome), and genomes of male mammals are faced with the challenge of pairing the largely nonhomologous X and Y chromosomes. It is not known whether and how these two prominent features—chromosome size and the presence of heteromorphic sex chromosomes—affect minimum DSB requirements.

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Separate but related to the question of why cells make a certain number of DSBs is how they ensure that the correct number is made. The DSB-forming machinery appears to be in excess over the DSBs actually formed (Neale et al. 2005; Milman et al. 2009; Lange et al. 2011), implying the existence of feedback mechanisms that influence SPO11 activity (Joyce et al. 2011; Lange et al. 2011; Zhang et al. 2011). However, it has been unclear how such feedback is integrated with meiotic chromosome dynamics.

To explore these facets of DSB control, we analyzed chromosome behaviors in mice that display an approximately twofold reduced meiotic DSB level. Our findings provide evidence that minimum DSB numbers are dictated principally by the pairing/synapsis requirements of the most vulnerable (smallest) chromosomes and that the presence of heteromorphic sex chromosomes affects the susceptibility of autosomes to pairing defects. Furthermore, we propose the presence of feedback mechanisms that integrate synapsis with the capacity of chromosomes to continue making DSBs.