Meiotic checkpoint

1.3.1. Mec1 and Tel1

S. cerevisiae Tel1 (telomere maintenance) and Mec1 (mitosis entry checkpoint) are orthologues of the mammalian signal transduction proteins ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia- and Rad3-related), which are also found in A. thaliana (ATM and ATR), C. elegans (ATM-1 and ATL-1), D. melanogaster (Tefu and Mei-41) and S. pombe (Tel1 and Rad3) (Carballo and Cha, 2007, Navadgi-Patil and Burgers, 2011, Phadnis et al., 2011). These chromosome-associated proteins play crucial roles in DNA damage and replication stress responses (Nyberg et al., 2002), as well as in unchallenged cell cycle in processes such as DNA replication and meiotic IH bias (Carballo et al., 2008, Cha and Kleckner, 2002, Hashash et al., 2011). Their inactivation confers sensitivity to genotoxic agents and genome instability (Novak et al., 2001, Zhou and Elledge, 2000).

Activation of Mec1/ATR and Tel1/ATM is mediated by ssDNA, coated by RPA and the 9-1-1 checkpoint clamp. In budding yeast, the clamp is composed of Ddc1, Rad17 and Mec3 (Hong and Roeder, 2002, Navadgi-Patil and Burgers, 2011). Interaction of the C-terminus tail of Ddc1 with Mec1 leads to the activation of the kinase domain of the latter (Navadgi-Patil and Burgers, 2009). Mec1 forms a heterodimer complex with Ddc2, the orthologue of human ATRIP (ATR interacting protein), which regulates its DNA binding affinity and, consequently, its kinase activity (Navadgi-Patil and Burgers, 2011).

Mec1/Tel1 kinase activity in yeast leads to phosphorylation of many targets, including mediator proteins like Rad9, which then leads to the activation of effector kinases, such as the key proteins Chk1 and Rad53 (orthologue of human Chk2) (Navadgi-Patil and Burgers, 2011). The consensus target sequences for these proteins consist of serine or threonine residues followed by glutamine residues (SQ/TQs). The presence of three or more of these motifs within 100 amino acids is

32

referred as a SQ/TQ cluster domain (SCD) and constitutes a hallmark for Mec1/Tel1 (ATR/ATM) targets (Traven and Heierhorst, 2005).

1.3.2. Hop1 and Mek1

Rad9 and Rad53 are two major targets of Mec1 (Section 1.3.1). Rad9 phosphoprotein contains multiple SQ/TQ sites, which, upon Mec1-dependent phosphorylation, allow its interaction with Rad53 and phosphorylation of the latter by Mec1/Tel1. A Rad53 transphosphorylation cascade mediated by Rad9 (Navadgi-Patil and Burgers, 2011, Usui et al., 2009) results in the full activation of Rad53 kinase. This leads to the phosphorylation of many downstream targets, important in the regulation of several cellular processes, such as DNA metabolism and checkpoint (Friedel et al., 2009).

During meiosis, checkpoint activation in response to defects in recombination (e.g., dmc1∆) or synapsis (e.g., zip1∆) is dependent on Hop1 and Mek1, but not on Rad9 and Rad53 (Carballo and Cha, 2007, Usui et al., 2001).

Indeed, Rad53 has been shown to be phosphorylated during meiosis in response to accidental breaks, but not SPO11-dependent DSBs, potentially a consequence of Rad53 being blocked from accessing the latter due to the altered meiotic chromosome axis structure (Cartagena-Lirola et al., 2008).

In fact, Hop1 and Mek1 proteins could be considered meiotic counterparts of Rad9 and Rad53, based on the following considerations (Carballo and Cha, 2007, Cartagena-Lirola et al., 2008):

i) Both Rad9 and Hop1 are Mec1 targets, being phosphorylated at SCD residues (Carballo et al., 2008, Sweeney et al., 2005);

ii) Rad53 and Mek1 are kinases and contain FHA (forkhead-associated) domains that are autotransphosphorylated, leading to kinase activation (Niu et al., 2009, Usui et al., 2009);

iii) Rad9-Rad53 interaction occurs via the phosphorylated SCD of Rad9 and the FHA domain of Rad53 (Usui et al., 2009). Hop1-Mek1 interaction also requires phosphorylation of Hop1 SCD and, particularly, of the threonine residue 318 in this domain (Carballo et al., 2008). FHA domains are known to interact with phosphorylated threonine residues (Durocher and Jackson, 2002), therefore suggesting that the mechanism of Hop1-Mek1 interaction is similar to that in their mitotic counterparts;

33

iv) Like Rad53, Mek1 acts as a Mec1 effector kinase (Niu et al., 2009, Rockmill and Roeder, 1991).

1.3.3. rad50S/sae2∆

In a rad50S or sae2∆ mutant, a delay in the exit of both meiotic divisions is observed (Hochwagen and Amon, 2006). In these backgrounds, DSBs are formed to normal levels, but Spo11 remains covalently attached to the DSB ends, blocking break resection and further processing, therefore leading to the accumulation of unresected DSBs (Alani et al., 1990, Prinz et al., 1997). This situation triggers a checkpoint response that requires Tel1 and Pch2 (Lydall et al., 1996, Usui et al., 2001). Since no ssDNA is generated in these mutants, recruitment of damage-sensing proteins to DSB sites requires factors that are unique to this checkpoint (Hochwagen and Amon, 2006).

Observations that mre11-58 single mutant, which also accumulates unprocessed breaks (Tsubouchi and Ogawa, 1998), and tel1∆ rad50S double mutant proceed through meiosis with no delays have led to the proposal that both Tel1 and MRX complex are sensors of protein-linked DSBs (Usui et al., 2001).

Recent data reveals that Pch2 and Tel1 act on a pathway that specifically signals unresected DSBs (Ho and Burgess, 2011). Furthermore, Pch2’s role in triggering rad50S/sae2∆ checkpoint was shown to require interaction with the N-terminus of Xrs2, consistent with the earlier hypothesis that Tel1 and the MRX complex are required for signalling of unresected DSBs.

Taken together, these observations suggest that the activation of rad50S/sae2∆ checkpoint relies on Tel1, Pch2 and the MRX complex. Additionally, the meiosis-specific structural proteins Red1, Hop1 and Mek1 are also required for arrest in rad50S/sae2∆ (Hochwagen and Amon, 2006, Longhese et al., 2008).

Activation of Mek1 kinase following interaction with phosphorylated Mec1 target Hop1 is also necessary, suggesting that Mek1 may take on roles typically associated to Rad53 during mitosis (Carballo et al., 2008, Niu et al., 2007, Wan et al., 2004, Woltering et al., 2000, Xu et al., 1997).

1.3.4. dmc1∆

As mentioned above, Dmc1 (disrupted meiotic cDNA) plays a crucial role in the formation of SEIs. In the absence of this protein, DNA is continuously resected, generating long segments of ssDNA (Bishop et al., 1992, Bishop, 1994). This

34

triggers a checkpoint response that is stronger than that observed in rad50S or sae2∆ mutants and results in meiosis arrest in prophase I (Bishop et al., 1992).

Arrest in dmc1∆ involves Rad24, the DNA damage sensor 9-1-1 and Mec1/Ddc2 complexes. Unlike in rad50S/sae2∆ checkpoint, Tel1 is not required (Hochwagen and Amon, 2006, Lydall et al., 1996). Hop1, along with Mek1 and Red1, is also required for this checkpoint response (Carballo et al., 2008, Eichinger and Jentsch, 2010, Niu et al., 2007, Wan et al., 2004, Woltering et al., 2000, Xu et al., 1997). However, the lack of checkpoint response observed in hop1 or mek1 mutants is due, not to the bypass of the checkpoint pathway, but to the inappropriate repair of DSBs by IS recombination (Carballo et al., 2008, Hollingsworth and Byers, 1989). Pch2 has also been shown to be required for dmc1∆ arrest in some yeast strain backgrounds (Borner et al., 2008).

1.3.5. zip1∆

Cell cycle progression is similarly arrested/delayed in the absence of synaptonemal complex components, such as Zip1, Zip2 and Zip3 (Hochwagen and Amon, 2006). Zip1 is the major component of the central element of the synaptonemal complex. In zip1∆ mutants, axial elements may line up side by side, but the distance between them is greater than that between lateral elements in a mature SC (Nag et al., 1995, Sym et al., 1993, Sym and Roeder, 1995).

The extent of cell cycle arrest in zip1∆ is variable, albeit generally less robust than in dmc1∆. It requires Mec1, Rad24, Rad17, Ddc1, Mec3, Hop1, Mek1 kinase and Red1 (Hochwagen and Amon, 2006, Roeder and Bailis, 2000). Pch2 has also been shown to be required for the synapsis checkpoint response in zip1∆

and zip2∆ mutants (Borner et al., 2008, Hochwagen and Amon, 2006, Shinohara et al., 2008).

1.3.6. Meiotic checkpoint in higher eukaryotes

Checkpoint mechanisms monitoring meiotic DSB repair have been primarily studied in budding yeast, but can also be found in several other model organisms, including flies (Ghabrial and Schupbach, 1999), nematodes (Bhalla and Dernburg, 2005) and mice (Gartner et al., 2000).

Disruption of RAD51-like genes in Drosophila, spnA, spnB or spnD, leads to defects in the formation of the karyosome, a meiosis-specific structure in flies, and in eggshell patterning. These defects are suppressed in mutants lacking Mei-W68,

35

the SPO11 orthologue in flies. The observation of these defects is also dependent on the presence of Mei-41 and Chk2, orthologues of the budding yeast Mec1 and Rad53 (Abdu et al., 2002, Ghabrial and Schupbach, 1999).

In the female germline of C. elegans hermaphrodites, cells with defects in DSB repair are removed by apoptosis, which occurs at prophase I, requiring the orthologues of yeast Rad17 and Rad24, MRT-2 and HUS-1, respectively (Colaiacovo et al., 2003, Gartner et al., 2000, Romanienko and Camerini-Otero, 2000). The nematode orthologue of budding yeast Hop1, HIM-3, appears to be required for checkpoint as well, since oocytes lacking this protein are not eliminated by apoptosis, despite defects in synapsis (Alpi et al., 2003).

In M. musculus, some differences are detected in terms of meiotic prophase checkpoints. Spermatocytes of Rad50^S/S mice, for instance, do not suffer a permanent meiotic block, but are instead eliminated through apoptosis, leading to a depletion in mature spermatocytes in these mutants’ testes (Bender et al., 2002).

Although absence of DMC1 in mice produces a meiotic arrest as in yeast (Baudat et al., 2000), inactivation of ATM (the Tel1 orthologue in mammals) in Dmc1^-/- mice produces a phenotype different from that observed in S. cerevisiae, as it does not bypass arrest. This is possibly due to functions of ATM in DSB repair (Libby et al., 2002, Romanienko and Camerini-Otero, 2000). As in yeast, however, Spo11 inactivation leads to the bypass of arrest in Dmc1^-/- (Barchi et al., 2005, Di Giacomo et al., 2005, Reinholdt and Schimenti, 2005).