The fragmentation observed in atm lines largely results from SPO11 activity
Garcia et al. previously described the semisterile phenotype and meiotic prophase defects of the Arabidopsis atm-1 mutant. Although homologs do synapse, and reductional segregation occurs in this mutant, there is extensive fragmentation that is probably responsible for the partial sterility. Essentially the same phenotype is also observed in atm knock-out mice (Xu and Baltimore, 1996): synapsis is delayed, but still occurs, together with extensive fragmentation. Given the well-established role of ATM in DSB signaling, it has been suggested that this fragmentation may be caused by a defect in the signaling and/or processing of programmed meiotic DSBs. However, it is also possible that these breaks reflect the accumulated spontaneous somatic DNA damage that has persisted ‘unchecked’ in the absence of ATM. Here, we demonstrate that atm lines defective in SPO11, the endonuclease that induces meiotic breaks, display a nearly complete correction of the fragmentation phenotype, consistent with the idea that these breaks are largely to the result of programmed SPO11 activity. This suggests that ATM, in WT cells, plays an important role in the processing of programmed breaks, the signaling of their presence (presumably in order to slow progression through meiosis) or in both processes. The defect in RAD51 assembly in atm−/− mice during meiosis (Barlow et al., 1998) is consistent with the notion that ATM plays a role in the processing of programmed meiotic DSBs. Arabidopsis mutants defective in XRCC3, a RAD51 paralog that is probably involved in the processing, but not in the initiation, of meiotic recombination events, display a meiotic phenotype identical to that of atm, and identical in its suppression by spo11 (Bleuyard and White, 2004).
A possible role for ATR in meiosis?
In partially synapsed chromosomes of mouse spermatocytes, ATR foci are associated with non-synapsed regions, whereas ATM clusters along synapsed regions (Keegan et al., 1996). However, the function of ATR in meiosis in higher eukaryotes is unclear, as atr mutants are lethal in animals (in yeast, the functions of ATR and ATM are to some degree fused into the single MEC1 protein, and this protein will be discussed later). As we reported earlier, the Arabidopsis ATR knock-out is not only viable, but is apparently fully fertile. Our analysis of meiotic stages from atr plants does suggest that there is a slight, possibly significant, increase in chromosomal fragmentation in atr, and that this is not suppressed by spo11. This fragmentation may result from the accumulation of undetected/unrepaired DNA damage in the atr line. Although it is not clear whether this fragmentation is meiosis specific, it does suggest that ATR plays some role in protecting genetic integrity in meiotic, as well as mitotic, cells. Of course, it is always possible that ATR plays a regular and important role in the process of meiosis, but that its failure to perform this role can be compensated for by another protein (such as ATM) that adequately copes with the consequences of this failure. This scenario is suggested by the extreme phenotype of the double mutant, described below.
Plants lacking both ATM and ATR display complete infertility and a strikingly defective meiotic phenotype
The Arabidopsis genome is distributed on only five chromosomes. Thus, in the complete failure of meiosis, where chromosomes distribute randomly, the odds of getting a complete chromosomal complement in the megaspore mother cell (with or without ‘extra’ chromosomes) is about 1 in 32. Given the fact that only pollen with a complete (or more than complete) genetic complement in the pollen-tube nucleus can germinate and grow to fertilize the megagametophyte, and that there is a great excess of pollen produced, we should expect to find two or three seeds per silique in the complete absence of organized meiotic segregation. This is approximately what we and others (Figure 3; Grelon et al., 2001) find in the spo11-1-1 mutant. These seeds suffer from poor germination (Table 1); however, this suggests that, although at least one copy of every chromosome must be present, there is significant aneuploidy.
In order to reduce fertility to zero, Arabidopsis must not only fail to simply segregate chromosomes properly, but must also fail to produce functional chromosomes. This appears to be the case for the atr atm double mutant.
Using atr and atm Arabidopsis mutants, we show here that ATR plays a partially overlapping role with ATM in the suppression of chromosomal fragmentation during meiosis. Although the atr single mutant does not have an obvious defect in meiosis, chromosomal fragmentation is significantly enhanced in the atr atm double mutant, in comparison with the atm single mutant. Interestingly, a defect in synapsis was observed in the double mutant. This defect was not complete: there appeared to be some pairing, but synapsis was never complete in any nucleus observed, and we do not know whether some or all pairing events were ectopic. This suggests that ATM and ATR might play a mechanistic role in early events in recombination, leading to a defect in homolog recognition. We emphasize that in both single mutants synapsis and the formation of bivalents was normal. Thus, this double mutant reveals a novel and essential role in synapsis that is performed redundantly by ATM and ATR.
As a consequence, perhaps, of ectopic annealing of non-homologous chromosomes, segregation at meiosis I was a complete failure. Although centromeres appeared to be pulled to the poles (Figures 1s,t and 2), the bulk of the chromatin remained at the metaphase plate in an entangled mass. The introduction of the spo11-1-1 mutation into the double mutant line corrected this aspect of the phenotype. In the triple mutant, chromosomes failed to interact and, although some fragmentation was observed, the univalent chromosomes were able to segregate at random. This indicates that the ectopic chromosomal interactions observed required the presence of SPO11-catalyzed breaks, and may have resulted from tentative and imprecise interactions between DNA sequences that, in the presence of ATM and/or ATR, would normally be corrected. However, other plausible hypotheses also fit these observations.
A role for the PI3K-like protein kinases in the homology search is supported by a comparison of the phenotype of the double mutant with that of other Arabidopsis mutants defective in end processing or single-strand introgression, including rad51, rad51c and mre11 (Li et al., 2004, 2005; Puizina et al., 2004). Each of these display a failure of synapsis (like the atr atm double mutant, but not the single mutants), chromosomal fragmentation and the (less severe) formation of anaphase bridges, as well as a correction of the fragmentation and bridge formation by spo11-1-1. In this study, we directly compare the phenotypes of the recombination-deficient mutant rad51 with atr atm, and show that there are major differences between the extents of ectopic chromosomal interactions, which are quite severe in the PI3K-like protein kinase double mutant. Similarly, S. cerevisiae mutants defective in MEC1, a PI3K-like protein kinase, combines many of the functions of ATM and ATR, display extensive meiotic ectopic recombination and ‘clumping’ of meiotic chromosomes into a single protein-dense mass, as do mutants defective in the checkpoint genes RAD17 and RAD24 (Grushcow et al., 1999). Thus, in yeast and plants, PI3K-like protein kinases act to prevent indiscriminant interactions between meiotic chromosomes. Although the effects of atr deficiency on mammalian meiosis have not been tested (because of the immediate lethality of the defect), ATR has been observed to coat non-synapsed regions of chromosomes (Perera et al., 2004). It also coats the sex chromosomes, in which programmed breaks occur but do not lead to homologous interactions. These observations are consistent with the notion that ATR, in mammals as well as plants, prevents sustained ectopic interactions between meiotic chromosomes. The suppression of these ectopic interactions by spo11-1-1 indicates that they require the induction of DSBs for their formation. It would be interesting to determine whether these interactions are RAD51 mediated, and perhaps represent strand-introgression events that require PI3K-like kinase activity for their reversal.
Activation of ATM is normally associated with activation of the MRE11/RAD50/NBS1 (M/R/N) complex, in which NBS1 is required for the signal transduction activity of the complex (D’Amours and Jackson, 2002; Falck et al., 2005). The plant homolog of NBS1 has recently been identified (Akutsu et al., 2007), and its biological significance, including a possible role in meiosis, has been investigated (Waterworth et al., 2007). Waterworth et al. established that the Arabidopsis NBS1 homolog is essential for cross-link repair (demonstrating its functionality), but is not required for meiosis. Interestingly, the atm nbs1 double mutant, like atm atr, reduced the fertility of the atm mutant to zero. The double mutant displayed a defect in synapsis, extensive fragmentation and clumping of the chromosomes: the same phenotype we observed here in atm atr. These data suggest that, in meiosis in plants, NBS1 is required for ATR function. Importantly, Waterworth et al.’s data indicated that NBS1 acts in the absence of ATM. This is quite a novel result, as NBS1 has heretofore been exclusively associated with ATM-dependent signaling. In certain contexts, ATM has been shown to activate ATR, and this activation requires NBS1 (Jazayeri et al., 2006), but in the case of plant meiosis, NBS1 and ATR may be interacting in the absence of mediation by ATM.
In summary, ATR and ATM play central roles in the response to DNA damage in mitotic cells, phosphorylating (in many cases) overlapping targets to initiate cell-cycle arrest, DNA repair, transcriptional regulation and, in animals, apoptosis. Although it is unclear whether ATR and ATM play analogous roles in the processing of DSBs during meiosis, our data indicate that both ATR and ATM can, under some circumstances, participate in the processing of SPO11-dependent breaks, and that the proteins act redundantly to prevent SPO11-dependent persistent ectopic interactions. Although ATR may simply ‘fill in’ for ATM in its absence, it is also possible that ATR plays either a truly redundant role with ATM, or a more specialized role in the processing of a particular and relatively rare subclass of breaks, as is suggested by the distinct contributions of the two kinases to mitotic γ-H2AX focus formation (Friesner et al., 2005).