Meiotic DNA double-strand breaks and chromosome asynapsis in mice are monitored by distinct HORMAD2-independent and -dependent mechanisms

Abstract

Meiotic crossover formation involves the repair of programmed DNA double-strand breaks (DSBs) and synaptonemal complex (SC) formation. Completion of these processes must precede the meiotic divisions in order to avoid chromosome abnormalities in gametes. Enduring key questions in meiosis have been how meiotic progression and crossover formation are coordinated, whether inappropriate asynapsis is monitored, and whether asynapsis elicits prophase arrest via mechanisms that are distinct from the surveillance of unrepaired DNA DSBs. We disrupted the meiosis-specific mouse HORMAD2 (Hop1, Rev7, and Mad2 domain 2) protein, which preferentially associates with unsynapsed chromosome axes. We show that HORMAD2 is required for the accumulation of the checkpoint kinase ATR along unsynapsed axes, but not at DNA DSBs or on DNA DSBassociated chromatin loops. Consistent with the hypothesis that ATR activity on chromatin plays important roles in the quality control of meiotic prophase, HORMAD2 is required for the elimination of the asynaptic Spo11 -/-, but not the asynaptic and DSB repair-defective Dmc1 -/-oocytes. Our observations strongly suggest that HORMAD2-dependent recruitment of ATR to unsynapsed chromosome axes constitutes a mechanism for the surveillance of asynapsis. Thus, we provide convincing evidence for the existence of a distinct asynapsis surveillance mechanism that safeguards the ploidy of the mammalian germline.[Keywords: asynapsis surveillance; meiotic prophase checkpoint; ATR; HORMA domain; meiosis; aneuploidy] Supplemental material is available for this article. Received January 30, 2012; revised version accepted March 19, 2012. Orderly chromosome segregation during the first meiotic division requires that homologous maternal and paternal chromosomes (called homologs) become physically linked, thereby forming so-called bivalents during the first meiotic prophase (Page and Hawley 2003). In most organisms, including mammals, meiotic recombination generates reciprocal exchanges, called crossovers (COs), between homologous DNA sequences. Interhomolog COs and sister chromatid cohesion together form the basis of the physical linkages, called chiasmata, that connect pairs of homologs during the first meiotic metaphase. Therefore, at least one CO must form between each pair of homologs to ensure correct segregation during the first meiotic division.CO formation requires that homologs find each other. To achieve this, double-strand breaks (DSBs) are actively introduced into the genome at the beginning of prophase by the SPO11 enzyme (Keeney et al. 1997;Baudat et al. 2000;Romanienko and Camerini-Otero 2000). This is followed by the resection of DSB ends, which creates 39 ssDNA overhangs (Neale et al. 2005). The RAD51 and DMC1 recombinases bind single-stranded DSB ends and assist homology search through promoting strand invasion of resected DSB ends into homologous DNA sequences (Baudat and de Massy 2007). Several DSB ends work in parallel on each pair of homologs to ensure alignment along the full lengths of homolo...

ATR acts stage specifically to regulate multiple aspects of mammalian meiotic silencing

“…To examine the role of ATR in silencing, we studied expression of the X-linked gene Scml2 in Atr flox/À male pachytene cells. Scml2 is silenced by MSCI, and mice defective in MSCI show inappropriate Scml2 expression at early pachytene (Wojtasz et al 2012). We observed Scml2 expression in 97% of Atr flox/À early pachytene cells, easily identifiable by their characteristic PAR gH2AFX staining (Fig.…”

“…The AE component synaptonemal complex protein 3 (SYCP3) (Kouznetsova et al 2009), HORMA (Hop1, Rev7, and Mad2) domain proteins HORMAD1 (Daniel et al 2011) and HORMAD2 (Wojtasz et al 2012), and breast cancer I gene BRCA1 (Turner et al 2004) have been identified as sensors: BRCA1 accumulates along unsynapsed AEs in an SYCP3-, HORMAD1-, and HORMAD2-dependent manner, and mice deficient in any of these four proteins exhibit MSCI defects. In contrast, the mediator of DNA damage checkpoint 1 (MDC1) (Ichijima et al 2011) and histone variant H2AFX (Fernandez-Capetillo et al 2003) are silencing effectors: In MDC1-and H2AFX-nulls, gene silencing within the chromosome loops does not occur.…”

Double-strand break repair on sex chromosomes: challenges during male meiotic prophase

“…It has also been suggested that components of the axial elements of synaptonemal complex including SYCP2 and SYCP3 might regulate the activities of recombination proteins to favor inter-homolog recombination. 10 Recent studies suggest that HORMA domain-containing (HORMAD) proteins regulate the interaction between homologous chromosomes, [11][12][13][14][15] which might also facilitate the usage of homologous chromosomes as templates for DNA repair and contribute to the inter-homolog bias in mammals.…”

“…52 Previous studies have suggested that HORMAD proteins are also important for proper localization of DNA repair proteins at unsynapsed axes. [11][12][13][14][15] Although the localization of HORMAD proteins is unaltered, the phosphorylation of HORMAD proteins is suppressed when ATR is depleted. 52 This observation suggests that some DNA repair proteins might be recruited to unsynapsed axes through recognition of HORMAD phosphorylation.…”

Clamping down on mammalian meiosis

“…5,14,15,[33][34][35]76,83 A variety of meiotic defects have been shown to lead to cell cycle arrest and apoptosis between zygonema and midpachynema in stage IV of the seminiferous epithelium. 77 These include cases of complete absence of DSBs (such as in Spo11 mutants), 88 impaired DSB repair (such as in Dmc1 and Msh5 mutants), [89][90][91] autosomal asynapsis, 92,93 or impaired MSCI. 94 This period has been loosely termed the pachytene checkpoint in mammals, despite being less well defined than in lower eukaryotes.…”