Meiosis is a specialized form of cell division involving one round of replication followed by two rounds of cell division, thereby reducing chromosomal content to produce haploid gametes. One hallmark process of meiotic cells is the recombination that occurs between homologous chromosomes during the prophase of the first meiotic division. Sites of meiotic recombination are initiated by the formation of DNA double-strand breaks (DSBs) (Sun et al., 1989). In yeasts, plants and most animals this activity is mediated by a topoisomerase-like endonuclease, which is termed SPO11 (Keeney and Neale, 2006; Keeney et al., 1997). Of the many SPO11-dependent DSBs that are formed (Moens et al., 2002), only a select few result in the exchange of chromosome arms that is required, in most organisms (Page and Hawley, 2003), for reductional division. However, all SPO11-generated DSBs must be repaired through homologous recombination to prevent mutagenesis and chromosome fragmentation.
Studies in yeasts and animals suggest that the molecular events leading to SPO11-dependent homologous recombination in meiotic cells are analogous to DSB repair in mitotic cells (Cromie et al., 2006; Haber et al., 2004; Hunter and Kleckner, 2001; Merker et al., 2003). Accordingly, most mutants defective in the repair of DSBs [via homologous recombination (HR) pathways] also display fertility defects. Models for homology-dependent mitotic DSB repair by homologous recombination are continually refined and expanded, as we gain further insight into the molecular players and their biochemical activities (Haber et al., 2004). Although there are multiple pathways for homology-dependent (RAD51-dependent) repair, all exhibit a requirement for the MRE11–RAD50–NBS1 (MRN) complex in 5′ end degradation, although the exonuclease activity of the complex itself is not required for this process. This hinge-like protein complex interacts with free chromosome ends, and activates a phosphoinositide-3 kinase (PI3K)-like protein kinase: termed ATM in animals, or MEC1 in yeast (MEC1 and, to a lesser degree, TEL1 activities in yeast perform the functions of both ATM and ATR in animals; see the discussion below). ATM (or MEC1) in turn activates the 5′ exonuclease activity fostered by the MRN complex, producing the free 3′ ends required for homologous recombination. Thus, PI3K-kinase like protein kinases play a role not only in signaling the presence of mitotic DSBs, but also in activating their repair, in both yeasts and mammals (Abraham, 2001; Jazayeri et al., 2006).
The role of ATM in the maintenance of genomic stability in plants has been genetically characterized in both mitotic and meiotic cells. For example, atm mutants in plants are sensitive to ionizing radiation, and display reduced fertility. Analysis of the meiotic stages in Arabidopsis atm mutants showed fragmentation of chromosomes in the early prophase I, which probably explains the reduced fertility of the mutants (Garcia et al., 2003), with similar results having been observed in mammals (Barlow et al., 1997; Xu et al., 1996). However, it has not yet been determined (in mammals or plants) whether the meiotic fragmentation results from persisting programmed DSBs, or from some other source of damage.
In mammals, ATM and the related protein kinase ATR have been shown to associate along synapsed meiotic chromosomes (Keegan et al., 1996; Moens et al., 1999). Furthermore, ATM is required for γ-H2AX phosphorylation at sites of DSBs in meiotic cells (Bellani et al., 2005), whereas ATR has been shown to co-localize with γ-H2AX during the early prophase I (Perera et al., 2004). These studies are clearly consistent with a role for ATM in the processing of meiotic DSBs, but leave an open question as to the significance of the presence of ATR. Because null mutant alleles of ATR are lethal in animals, the role of ATR in processing DSBs during meiosis has been difficult to assess directly.
In addition to the roles of ATM and ATR in processing DSBs, both kinases are key players in mitotic cell-cycle arrest and apoptotic responses to DSBs (Abraham, 2001; Shiloh, 2003). It is possible that ATM and/or ATR could play similar roles during the meiotic cell cycle, monitoring the progression of DSB repair, before progressing into the next meiotic cell stage, as has been proposed previously (Barlow et al., 1998; Hochwagen and Amon, 2006). In support of this, mutants of MEC1, the ATM/ATR homolog in Saccharomyces cerevisiae, progress prematurely into the first meiotic division in the presence of unrepaired DSBs, unlike wild-type (WT) cells (Lydall et al., 1996). However, the ATM-dependent damage response pathway is not essential for meiotic arrest or apoptosis responses, as ATM-deficient mice themselves display meiotic arrest and apoptotic degeneration early in prophase I (Barlow et al., 1997; Xu et al., 1996), as well as chromosomal fragmentation. One possibility is that ATR-dependent pathways can also regulate meiotic progression.
Plants display neither apoptosis nor a terminal meiotic arrest in response to unrepaired DSBs; various homologous recombination mutants of Arabidopsis, such as dmc1 (Couteau et al., 1999) and rad51 (Li et al., 2004), the latter of which displays an abundance of chromosomal fragmentation in early prophase I, complete meiosis. However, these studies did not measure the rate of progression of the cell cycle, and it is possible that a transient arrest occurs. Because Arabidopsis atr single and atr atm double mutants are viable (Culligan et al., 2004), Arabidopsis provides a valuable model system for studying the role of ATR and ATM during the upstream meiotic recombination process.
Employing null Arabidopsis ATR and ATM mutants, we show here that both ATR and ATM can contribute to the repair of SPO11-dependent DSBs. Although the atr single mutant does not display meiotic defects, the atr mutation in the atm background compounded the fragmentation observed in the single atm mutant, suggesting a partially redundant role for ATR and ATM during meiosis. Mutations inducing defects in SPO11 reversed both the fragmentation and the extensive ectopic interactions observed in the atm single and atr atm double mutant lines. These results suggest that ATM (and possibly ATR) facilitate the timely processing of SPO11-dependent DSBs during meiosis, and that both proteins play a critical role in preventing persistent and indiscriminant interactions between chromosomes during meiosis.