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Available online at www.sciencedirect.com

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Review Article

Homeostatic regulation of meiotic DSB formation by ATM/ATR Tim J. Cooper, Kayleigh Wardell, Valerie Garcia, Matthew J. Nealen Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK

article information

abstract

Article Chronology:

Ataxia–telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central

Received 18 June 2014

players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks

Accepted 14 July 2014

(DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle

Available online 27 July 2014

control to damage-sensing and repair processes in order to prevent untimely cell cycle progression

Keywords:

while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the

ATM

process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes

ATR

via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is

Mec1

the introduction of numerous DSBs spread across the genome by the evolutionarily conserved enzyme,

Tel1

Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-

Meiosis

DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.

Spo11

& 2014 Elsevier Inc. All rights reserved.

Recombination

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Meiotic checkpoints and signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Positive regulation of DSB formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Negative regulation of DSB formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 ATM-dependent regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Trans-inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 DSB interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 ATM/ATR targets - Spo11 accessory factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 Mechanisms of homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Author contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

n

Corresponding author. fax: þ44 1273 678121 E-mail address: [email protected] (M.J. Neale).

http://dx.doi.org/10.1016/j.yexcr.2014.07.016 0014-4827/& 2014 Elsevier Inc. All rights reserved.

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Introduction Throughout early prophase I, meiotic cells intentionally generate DSBs as part of a stringently controlled process to initiate homologous recombination (HR). HR is a critical DSB repair pathway that ultimately permits the exchange of genetic information between homologous chromosomes (homologues) and which is essential for reductional chromosome segregation during the first meiotic nuclear division. Saccharomyces cerevisiae, within which much of the molecular detail of HR has been elucidated, employs the meiosis-specific and evolutionarily conserved type-II topoisomerase-like enzyme, Spo11, to generate DSBs [2]. DSB formation is accomplished via a Spo11-catalysed transesterification reaction that generates covalent protein-DNA intermediates— whereby Spo11 monomers remain linked to the newly created 50 termini. Spo11 and DSB formation additionally require the concerted efforts of nine other factors including Mre11, Rad50, Xrs2/ Nbs1, Sae2, Rec114, Mer2 and Mei4. Mre11, Rad50, Xrs2 and Sae2 coordinate removal of Spo11-intermediates via endonucleolytic cleavage, releasing Spo11-oligonucleotides. Subsequent exonucleolytic resection generates 30 ssDNA tails—the prime substrates for HR [3]. These ssDNA tails, aided by specialised recombinases, invade complementary regions on homologues generating interhomologue interactions. In many organisms, inter-homologue interactions drive the physical synapsis of the involved chromosomes via formation of a proteinaceous “zipper” designated the synaptonemal complex (SC) [4]. Given that DNA damage resides at the heart of meiosis, it is unsurprising to find that ATM/ATR and respective orthologues feature prominently in the meiotic landscape. To date, ATM/ATR and their downstream effectors have been implicated in a wide range of meiotic events including promotion of HR at various steps, repair-template choice and DSB repair, control of crossover formation and distribution, synapsis checkpoints and homolog pairing, meiotic chromosomal segregation, X-chromosome inactivation and sex body formation. Such work, reviewed extensively in [5], has revealed that not only have traditional ATM/ATR cell cycle surveillance mechanisms been co-opted for meiosis but that the kinases are additionally involved in uniquely meiotic processes and have evolved meiosis-specific targets. Interestingly, while the number of DSBs typically formed per meiotic cycle differs between species, such differences do not significantly scale with genome size [6–11]. Moreover, DSB frequency is maintained at a moderate level despite an apparent excess of Spo11 protein [12], hinting at strict regulatory control. This phenomenon, termed DSB homeostasis [13,14], is proposed to maintain levels of DSBs within genetically-encoded ranges in order to prevent the deleterious effects associated with too few or too many DSBs [10,15,16]. Several recent developments have strongly implicated ATM/ATR-dependent systems as being integral to DSB homeostasis and the regulation of DSB formation. In this review we will thus explore the mechanisms underpinning these two inter-related aspects of ATM/ATR meiotic function and provide an overall framework for meiotic DSB homeostasis.

Meiotic checkpoints and signalling Akin to mitotic cycles, checkpoint mechanisms exist within meiosis. The pachytene-checkpoint, operating during prophase I,

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surveys the status of DSB-repair and homolog-synapsis in order to arrest cells until such processes are completed [5,17]. Given that premature anaphase I entry proves lethal, this checkpoint is of critical importance [18]. Transmission of pachytene checkpoint signals primarily depends upon the ssDNA-sensing ATR system comprising ATR, the RAD9–RAD1–HUS1 (9–1–1) clamp complex and the RAD17 clamp-loader; respectively designated Mec1, Rad17, Mec3, Ddc1 and Rad24 in S. cerevisiae [18–20]. A central target of the checkpoint in S. cerevisiae is Ndt80, a meiosis-specific transcription factor responsible for exit from pachytene into anaphase via the induction of key genes involved in cell cycle progression and Holliday-junction resolution [21–23]. Checkpoint signals inhibit Ndt80 via suppression of its hyper-phosphorylation —a modification required for its transcription factor activity— ultimately arresting cells within prophase I [24] [Fig. 1]. In addition to this, a mitotic-like replication checkpoint also appears to be active in pre-meiotic S-phase within S. cerevisiae and requires Mec1-signalling [25]. ATM (Tel1 in S. cerevisiae) primarily signals via CHK2 and is recruited to DSB ends via the Mre11– Rad50–Xrs1/Nbs1 complex (MRX/N), whose Mre11 subunit exhibits direct DSB-binding activity [1]. The pachytene checkpoint also relies upon ATM signalling, albeit to a lesser extent [5]. A meiosis-specific paralogue of CHK2, known as Mek1, has been identified within S. cerevisiae [26,27]. Both Mec1 and Tel1 signals feed into Mek1 activation during meiosis, integrating multiple stimuli into a common target [27–29]. Ablation of Mek1 activity reduces the viability of S. cerevisiae spores (the haploid products of yeast meiosis) suggesting that Mek1 is a major effector of Mec1/Tel1 meiotic activity [26,30]. In some lab strains of S. cerevisiae, prophase arrest is also regulated by the evolutionarily conserved hexameric ATPase, Pch2 (TRIP13 in mammals). Pch2/ TRIP13 appears to act by promoting the remodelling of the HORMA domain-containing meiotic chromosome component Hop1 (HORMAD1/2 in mammals), which are targets of the ATM/ ATR response, thereby aiding prophase arrest in response to defects in chromosome pairing and synapsis [29,31]. Despite sharing a subset of downstream effectors and targeting identical motifs (SQ/TQ sites) [1], ATM and ATR appear to possess distinct roles during meiosis as explored in the following sections.

Positive regulation of DSB formation Recent work within S. cerevisiae has revealed a Mec1/Rad24/ Rad17-dependent positive feedback loop that promotes DSB formation under conditions of suboptimal DSB catalysis [15,32]. Specifically, strains carrying hypomorphic forms of Spo11 (spo11HA and spo11-D290A) or mutation of the PCH2 gene display significantly reduced meiotic DSB formation when Spo11 removal and ssDNA resection are blocked. By contrast, only minor reductions in DSB frequencies and spore viability are observed in cells capable of removing Spo11 from DSB ends to expose ssDNA. This apparent ability of resection-proficient cells to compensate for reduced Spo11 activity is abolished when components of the ATRbranch of the checkpoint pathway (MEC1, RAD24 and RAD17) are mutated, resulting in severely reduced DSB frequencies and synergistic reductions in spore viability despite the fact that spo11-HA or spo11-D290A alone display no appreciable reduction [15,32]. Collectively, these results suggest that the transient formation of ssDNA at meiotic DSBs creates a signal—transduced

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Spo11 + Accessory Factors

Wildtype

Rad24/Rad17

Mec1

Rate of DSB Formation

DSB

Activation Threshold Spo11 (Hyp) + mec1

Spo11 (Hyp)

Spo11 (SuperHyp) Ndt80

P

Inactive Metaphase-I Entry

Cell-cycle progression

Delayed MI

Active

Time

Premature MI

P

Ndt80

Fig. 1 – Positive regulation of DSB formation within S. cerevisiae. Left, ssDNA at meiotic DSBs, produced via enzymatic resection, is detected by components of the DNA-damage response (DDR), namely Mec1(ATR), the Rad17–Mec3–Ddc1 clamp complex and the Rad24 clamp-loader. Transient activation of the DDR promotes further Spo11 DSB catalysis by inhibiting the hyperphosphorylation of Ndt80, resulting in transient cell cycle arrest and suppression of the negative effect that Ndt80 exerts over DSB formation. Right, Kinetic models of meiotic DSB formation in various mutants. Graph lines show the theoretical rates of DSB formation within each marked background. The activation threshold represents the point at which Mec1 signal strength is sufficient to implement Ndt80 inhibition to arrest prophase I. Both super-hypomorphic spo11 strains and the spo11-hyp/mec1Δ mutants exit prophase prematurely due to a failure or inability to activate the DDR, and therefore a failure to inactivate Ndt80. Weaker Spo11 hypomorphs activate sufficient Mec1 signalling to prolong prophase I, exiting at a later time compared to wildtype. See in-text for further details. Abbreviations: Hyp¼ Hypomorph/SuperHyp ¼Super Hypomorph.

by Mec1/ATR—that promotes further Spo11-DSB catalysis within the cell. Importantly, deletion of Ndt80 within spo11-HA/D290A rad24Δ or rad17Δ backgrounds was found to significantly rescue these defects in DSB formation [15,32], and even moderate extension to meiotic prophase, mediated by transient depletion of Ndt80 activity, is sufficient to restore spore viability [15]. Given the pivotal role of Ndt80 in pachytene exit and its requirement for shutdown of Spo11-dependent DSB catalysis [33], these findings suggest that Mec1-dependent inhibition of Ndt80 results in sustained Spo11 activity and transient prophase I arrest [Fig. 1 left]. The latter could critically allocate the cell an extended period of time to generate and subsequently repair DSBs without interruption by unscheduled anaphase I entry. Consistent with this idea, Ndt80 deletion and/or transient depletion rescues defects in synapsis, recombination rate and spore viability within a set of extreme Spo11 hypomorphs. Surprisingly, such hypomorphs suffer these meiotic problems even in the presence of an active Mec1/ATR pathway [16]. The differing and perplexing behaviour of weak and strong Spo11 hypomorphs likely reflects the dynamic nature of signalling and DSB steady-state kinetics [Fig. 1 right]: (i) if DSB formation exceeds repair rate, as within the early stages of wildtype prophase, sustained checkpoint activation will occur—prolonging prophase I to permit sufficient DSB formation and repair. Consistent with this, permanent arrest occurs in dmc1Δ repair-defective strains within which DDR-activating intermediates of HR persist [34]; (ii) if DSB formation rate equals

repair rate, as within weak Spo11 hypomorphs, a transient and low amplitude DDR may occur that may prove sufficient to regulate the process provided DSB formation and/or DDR signals overlap; (iii) if DSB repair rate significantly exceeds DSB formation, any threshold for DDR activation may not be reached, uncoupling repair status from cell cycle state. Under these latter conditions the cell will proceed to activate Ndt80, subsequent cyclin expression and anaphase I entry regardless of whether sufficient DSBs have been generated. Indeed, catalytically-dead spo11-Y135F mutants progress through to anaphase I more quickly relative to wildtype [35]. Spo11-hypomorphs that cause severe defects, even in the presence of functional Mec1/Rad24/Rad17 signalling, may thus simply fail to cross the activation threshold and enter a state where they are unable to compensate for lower catalysis rates—a state highly representative of scenario (iii) and one that still could be partially rescued by synthetic inactivation of Ndt80. An important question is: If extension of prophase I seemingly bypasses reliance upon checkpoint signalling—why the reliance and complexity to begin with? This undoubtedly reflects the need for a robust and adaptive system to deal with the complexities of meiosis—a concept further expanded upon below. A prediction of this regulatory model would be that DDR-deficient strains would mimic spo11-Y135F catalytically-inactive mutants and result in quicker meiotic transit. Contrary to this, rad17Δ or rad24Δ mutation slows progression relative to wildtype as assayed by delayed chromosome segregation at anaphase I [35]. rad17Δ or rad24Δ mutations, however, decrease colocalisations of the Rad51

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and Dmc1 recombinases, result in hyper-resection of DSBs, and increase levels of non-allelic recombination hinting at mispairing of non-homologous chromosomes [15,35]. Thus the apparent delay in meiotic progression within DDR mutants may be due to mechanical issues pairing and segregating chromosomes during metaphase I rather than due to a delayed exit from prophase I, which we propose most likely still occurs prematurely. While these observations provide an attractive model for the positive regulation of DSB formation, contradictory data suggesting Rad17 is a negative regulator have also been reported [32]. Furthermore, the synergistic effect of a Spo11-hypomorph within a rad17Δ background is less pronounced than within mec1Δ or rad24Δ, resulting in a smaller reduction in DSB formation [15]. Taken together, these observations raise the possibility that Rad17 mediates both positive and negative regulation in Mec1/Rad24dependent and Mec1/Rad24-independent manners respectively. Deletion of Rad17 could thus eradicate a positive effect, producing a synergistic reduction in DSB frequency that is stunted due to the removal of a negative effect. Tel1/ATM has also been implicated as a positive regulator whose deletion results in lowered DSB frequencies [25,32]. This is a striking find considering that Tel1/ ATM has a more generally accepted role in negative regulation as discussed below. Such opposing observations highlight the complexity of the homeostatic system under consideration.

Negative regulation of DSB formation ATM-dependent regulation ATM is primarily implicated in the negative regulation of DSB formation and key examples of this have been recently observed within M. Musculus, S. cerevisiae and D. melanogaster model systems. DSB formation during prophase I, as ascertained by assessing Spo11-oligonucleotide levels, is reported to significantly increase within Atm  /  -null mice compared to wild-type [10]. It is known that Atm  /  mice display severe meiotic defects that ultimately result in infertility [36–38]. In a striking contrast to the loss of Mec1 activity in S. cerevisiae, which displays a synergistic defect with Spo11 hypomorphs (see above), reduction in DSB formation via means of Spo11 heterozygosity largely rescues the defects normally observed in Atm  /  mice—attributing the Atm  /  meiotic phenotype to the excessive formation of DSBs [10]. Consistent with the idea of increased DSB formation, crossover frequencies are also increased within Atm  /  mice [39]. Interestingly, ataxia telangiectasia patients, who contain a mutated form of ATM, also display infertility—hinting that ATM may play a similar meiotic role within humans [40]. Comparable mechanisms also appear to function within Drosophila and S. cerevisiae. Inactivation of the Drosophila ATM homologue, tefu, results in substantial increases in g-H2AV foci formation within both nurse cells and oocytes [8]. As g-H2AV is a functional homolog of mammalian g-H2AX, which forms in response to DSBs, these observations directly implicate tefu within the negative regulation of DSB formation [8,41]. The studies conducted within S. cerevisiae have revealed extra layers of complexity and molecular detail to this seemingly conserved mechanism of ATMdependent negative regulation (discussed below). In addition to its role in positive regulation, Mec1 has also been paradoxically shown to mediate specific branches of negative regulation in S. cerevisiae. The following subsections focus on these findings from the yeast model system.

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Trans-inhibition Within S. cerevisiae, wild-type distributions of DSBs, as inferred by post-meiotic analysis of recombination, are not compatible with models describing independent distributions of DSBs across the four chromatids that are eventually held within each synapsed chromosome [42]. Instead, it appears formation of a DSB on one chromatid suppresses formation on another at the corresponding locus. In many instances, such inhibition appears to affect all three partner chromatids such that only one DSB forms per chromatid quartet at any given locus [42] [Fig. 2 left]. This phenomenon is known as trans-inhibition and it may serve to ensure that an intact repair substrate is always available while preventing potentially deleterious double recombination events from occurring [42–44]. Interestingly, Tel1 or Mec1 mutants severely reduce the occurrence of one specific type of transinhibition—however it is not yet clear whether this is interhomolog or inter-sister trans-inhibition, or if the same type is being lost in both mutants. The mechanisms of trans-inhibition have not been explicitly investigated; however, recent data raise the possibility that the process is HR-dependent. In vegetatively growing S. cerevisiae cells, induction of a DSB by means of a SceI cleavage site, not only results in loading of Rad51 and H2A phosphorylation (a Mec1/Tel1 target) in the vicinity of the break, but also at discrete locations across all chromosomes [45,46]. This infers that ssDNA molecules, engaging in homology searching, can “deliver” biological activity to other chromosomes—an ability that sits well with the requirements of trans-inhibition. ssDNA may thus provide a platform for, or trigger an event that results in, Mec1/Tel1 activation, with the highest levels of activation occurring once full homology has been found. Such a mechanism has the potential to mediate trans-inhibition of meiotic DSB formation specifically between corresponding allelic loci on homologous chromosomes. By contrast, given the spatial proximity of sister chromatids, local pools of activated Mec1/Tel1 may be sufficient to mediate inter-sister trans-inhibition without a dependency upon downstream HR steps. One alternative possibility is that Mec1 and Tel1 mediate opposing forms of trans-inhibition. For example, given the potential role of ssDNA within HR-dependent inter-homolog trans-inhibition, this form of inhibition may exclusively rely upon the ssDNA-sensing Mec1. On the other hand, Tel1 may be restricted to mediating trans-inhibition on a more local scale (inter-sister), a behaviour that closely resembles one of Tel1's major functions (see below).

DSB interference As with meiotic recombination events between chromatids, events along a chromatid also exhibit non-random patterns— most frequently noted in the distributions of chiasmata and genetic crossovers (crossover interference) [47,48]. The mechanisms mediating crossover interference are, however, layered upon the preceding and precursor decision: the distribution of DSBs, a highly regulated process for which mechanisms are now beginning to be elucidated. Our recent work in S. cerevisiae has unveiled the existence of a novel Tel1-dependent process influencing DSB distribution: DSB interference [66]. We use the term DSB interference to describe the in-cis suppression of DSB formation— whereby the coincident formation of adjacent DSBs occurs less often than expected by chance. Tel1 inactivation results in a loss of DSB interference, allowing DSBs in adjacent regions to arise independently of one another. Interference presumably occurs

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Axis

Interhomolog trans-inhibition Mec1/Tel1 HR? Interference

Sister Chromatids

Homologous Chromosomes

Chromosomal Loops

Tel1

Stalled Replication Fork

DDK Intra-loop Interference Mer2 Mei4 Rec114

Mec1/Tel1

Mec1 Rad17-Mec3-Ddc1 Rad24

P

Intersister trans-inhibition

RPA

1

2 3

Spo11

Double-strand break

DSB Formation

DSB hotspot

Fig. 2 – Negative regulation of meiotic DSB formation within S. cerevisiae. Left, Meiotic chromosomes are organised into linear arrays of loops with sister chromatid paired by chromosomal axes. Inhibition can occur in trans, whereby a DSB inhibits DSB formation at a corresponding locus on either the sister chromatid or the homologous chromosome. Trans-inhibition may involve Tel(ATM) and/or Mec1(ATR), and may depend on active HR (see in-text). Additionally, inhibition can occur in cis in a Tel1dependent manner within loops and across flanking regions. It is likely that ATM-dependent negative regulation within M. Musculus and D. melanogaster occurs via similar or identical mechanisms. Right, DSB replication and DSB formation are intimately coupled such that delayed replication causes a local delay in DSB formation. Inhibition of DSB formation is thought to be achieved via a number of mechanisms: (1) Inhibition of DDK-dependent phosphorylation of Mer2, preventing Spo11 recruitment; (2) Inhibition of Rec114 loading; (3) Inhibition of Spo11 at the level of transcription. See in-text for further details.

when, and only when, a DSB has formed at the test locus. A related but distinct phenomenon, known as DSB competition, has also been previously documented whereby insertion of a strong DSB hotspot suppresses break formation in adjacent regions [43,44,49,50]. However, in contrast to DSB interference, DSB competition appears to exhibit Tel1-independency (V. Garcia, R. M. Allison, and M.J. Neale; unpub. obs.) and may prove DSBindependent; solely relying upon alterations in chromatin structure to mediate its effects. Surprisingly, Tel1 appears to modulate DSB interference within the confines and context of the unique structures present on meiotic chromatin—the chromatin loops. Each meiotic chromatid contains dispersed association sites that dock onto a chromosomal axis while intervening sequences protrude outward, forming a linear array of loops which harbour the sites of DSB formation [51,52] [Fig. 2 left]. DSBs arising independently in the absence of Tel1 only do so when being sufficiently spatially separated so as to reside within separate loop domains. Contrastingly, for closely spaced DSBs, inactivation of Tel1 results in coincident DSB formation at rates higher than those expected by chance. Strikingly, this concerted DSB activity is restricted to  15 kb windows correlating strongly with singular loop domains, revealing a more pronounced intra-loop inhibitory effect orchestrated by Tel1 [Fig. 2 left]. Loops often contain several potential DSB sites, all of which may undergo “priming” by intimate association with Spo11 accessory factors. Our data ultimately suggest that Tel1 is required to ensure that only a single site per loop is utilised and that adjacent sites both within the loop and in the surrounding regions are then inhibited for DSB formation. The activity of Tel1 within DSB interference may also explain previously contradictory data (mentioned earlier) that suggested Tel1 actually

serves to promote DSB formation, specifically within larger chromosomes [25,32]. If inactivation of Tel1 indeed results in a gross redistribution and increase of DSB events on chromatids that are already cut at least once (as per our data), classical methods for quantifying DSB frequencies such as pulsed-field gel electrophoresis—which measure total broken chromatids rather than frequency of breaks per chromatid—are unlikely to be able to distinguish between this eventuality and legitimate reductions in DSB levels.

ATM/ATR targets - Spo11 accessory factors Spo11 accessory factors, a collective set of nine proteins essential for DSB formation, are emerging as central responders to ATM/ ATR signals. In particular, the Spo11-accessory factor Rec114, which coalesces with Mer2 and Mei4 into the RMM-complex and preferentially binds around axial sites, is a key target of both Tel1 and Mec1 within S. cerevisiae [13,53]. Rec114 undergoes Tel1/ Mec1- and DSB-dependent phosphorylation, and constitutively mimicking this phosphorylation reduces chromatin association as well as genome-wide DSB levels [13]. While it is not possible from these data to distinguish effects of Tel1 over Mec1, Rec114 phosphorylation represents one potential way in which ATM orthologues can impose negative regulation over DSB formation and also raises the possibility of Mec1 as a negative regulator. Interestingly, Ndt80 modestly contributes to Rec114 degradation [13]. Thus, Mec1's inhibition of Ndt80 (as discussed above) has the potential to prolong Rec114 presence, contributing to Mec1's positive effect over break formation. Outside the scope of DSB homeostasis, DSB formation is also tightly coupled to the state of pre-meiotic S phase. Previous data has reported an in-cis and local coupling between DNA replication

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and DSB formation whereby delayed replication of a chromosome arm delays break formation within the same region [54,55,67]. Such regulation is of the upmost importance: replication fork progression is fully stalled by DSBs and subsequent topological constraints can result in full fork collapse [56]. Mistimed and widespread induction of meiotic DSBs would thus prove fatal if DNA replication remained uncompleted. Within vegetatively growing cells the replication checkpoint, which depends upon ATR and the downstream CHK1, inhibits cell cycle progression in response to aberrant replication forks [1]. Several pathways of Mec1-dependent regulation have been uncovered recently which demonstrate that this checkpoint also functions within S. cerevisiae meiosis [25] [Fig. 2 right], feeding information forward to ensure DSB formation is appropriately timed and further illustrating the repurposing of existing abilities to sense damage during meiosis. Mec1/Rad53 appear to mimic their mitotic roles, limiting the activity of Dbf4-dependent Cdc7-kinase (DDK) within pre-meiotic cells treated with the replication inhibitor hydroxyurea (HU) [25]. As Mer2 requires DDK-dependent phosphorylation in order to recruit Spo11 to axial sites, the down-regulation of DDK by Mec1/Rad53 inhibits a crucial step in DSB formation in response to replication stress [25,57]. Inhibition of DNA replication also causes a 10-fold reduction in Spo11 transcript levels—a reduction partially ablated within mec1Δ mutants, implicating Mec1 within Spo11 down-regulation at the level of transcription via unknown mechanisms [25]. Furthermore, Mec1 activity appears to inhibit loading of Rec114 and Mre11 in response to replication stress [25]. Cells thus clearly employ a multifaceted approach to temporally regulate DSB formation in coordination with DNA replication.

Mechanisms of homeostasis At face value, homeostatic mechanisms may seem like nothing more than a simple balance between opposing forces; however, the ability of Mec1/Tel1 to each orchestrate both negative and positive regulation greatly complicates the system under consideration and argues against any form of linearity. The biological outcome of any signalling pathway can greatly vary in response to a number of things including the ways in which its components are spatially or temporally regulated, signal/stimulus strength, target abundance and phosphorylation efficiencies [58]. DSB homeostasis undoubtedly employs a similar array of methods to achieve balance between positive and negative regulation under multiple cellular contexts. As DSB formation is not reactivated as repair progresses and break levels fall, there must exist a point at which the influence of the discussed ATM/ATR-dependent mechanisms is diminished. Interestingly, homolog synapsis was recently shown to suppress DSB formation and represent a novel branch of regulation [59]. Thus, it may prove that the mechanisms discussed throughout constitute a first phase of regulation that ultimately ensures synapsis is achieved in those organisms whose synapsis is DSB-dependent. Consequently, the cell then enters the second phase where it effectively “hands-over” control to the mediators of synapsis-dependent regulation as well as Ndt80, priming the cell for chromosomal segregation. Indeed, formation of the synaptonemal complex as assayed via Zip1 (S. cerevisiae) and SYCP3 (mouse) staining, strongly correlates to loss of Hop1 and HORMAD1/2 respectively—loss that is dependent upon Pch2/

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TRIP13 [31,60,61]. Such observations highlight the ability of synapsis to remove a critical ATM/ATR substrate and its potential to initiate a “hand-over” between regulatory phases. The synaptonemal complex could thus be considered a “checkpoint” factor in its own right. However, whether or not synapsis-dependent feedback depends upon ATM/ATR is currently unknown and certainly cannot be ruled out.

Concluding remarks While detection of meiotic damage is assumingly identical to its mitotic counterpart, there has been an obvious adoption of meiosis-specific targets outside the scope of traditional ATM/ ATR targets. Evolution of a meiotic DDR and a system to carefully regulate DSB formation likely reflects (i) the absolute requirement for cells to be able to stringently manage widespread, potentially fatal damage; (ii) the need to homeostatically buffer biological variability on a cell-to-cell basis and (iii) the ultimate purpose of meiosis: the exchange of genetic information. Indeed, as evidenced here, both Mec1- and Tel1-dependent systems safeguard genomic integrity by regulating DSB formation in various manners ensuring that when DNA replication or anaphase I do occur, they proceed without error. Consistent with the requirement to buffer natural variation—polymorphisms, temperature and nutritional status have all been reported to impact recombination frequencies [62–65]. The observation that isogenic Spo11 hypomorphic S. cerevisiae strains produce wide ranges of recombination frequencies further illustrates the need for a foolproof system to buffer biological variability on a cell-to-cell basis even when an identical genetic complement is present [16]. The Mec1/Rad17/ Rad24 systems, at least in S. cerevisiae, clearly ensure that prophase I duration strictly matches the specific situation of the cell at hand; suppressing premature anaphase I entry but also preventing prophase I from running longer than is required, ensuring succession into the protective sporulated state is as fast as possible. Simultaneously, Tel1, alongside Mec1, regulate DSB distributions in such a way to allow fruitful and appropriate levels of genetic exchange without risk to cellular survival. Ultimately, the complexity of the homeostatic mechanism considered here underscores the complex and multifaceted nature of the meiotic process. DSB homeostasis has likely arisen, out of necessity, to be both robust and efficient, and despite its heavy evolutionary demand upon the pre-existing cellular pathways, it has remained largely conserved—contributing critical regulation to the highly advantageous process of sexual reproduction.

Author contributions TJC drafted the manuscript and figures. KW, VG and MJN provided comments and contributed intellectually during manuscript preparation.

Acknowledgments TJC, KW, and VG are supported by awards made to MJN: a Consolidator Grant from the European Research Council under Grant no. 311336; a University Research Fellowship from the

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Royal Society, and a Career Development Award from the Human Frontiers Science Program Organisation. [17]

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