Eroded telomeres tethered at the nuclear envelope
The tethering at the nuclear envelope of highly repeated genomic loci, such as the 9.1 Kb rDNA tandem repeats and the 300 bp arrays of telomeric TG1-3 repeats, plays an important role in preventing aberrant recombination events that could be detrimental to genome stability. Accordingly, hyper-recombination phenotypes were observed at eroded telomeres formed in absence of the ATM kinase Tel1, since they are not anymore anchored at the Nuclear Envelope by Mps3 (Schober et al., 2009).
Eroded telomeres in telomerase negative cells are localized at Nuclear Pores instead of being anchored at the nuclear envelope (Khadaroo et al., 2009). The relocation to nuclear pores depends on the SUMOylation by Siz1/Siz2 of the replication protein A (RPA), which is bound to resected telomeres and mediates the interaction of eroded telomeres with the SUMO targeted Ubiquitin ligase (STUbL) Slx5-Slx8 (Churikov et al., 2016), located at the NPC through association with Nup84 (Nagai et al., 2008). The interaction of eroded telomeres with Slx5-Slx8 at the nuclear pore triggers type II telomere recombination, during which TG1-3 repeats are amplified in a process dependent on Rad52 and Sgs1 (Churikov et al., 2016). Because Slx5-Slx8 interact with a subunit of the 26S proteasomal lid (Krogan et al., 2006), it has been speculated that the Slx5-Slx8-dependent nuclear localization of SUMOylated telomere bound proteins leads to their Ubiquitin-dependent proteasomal degradation. Indeed, Slx5-Slx8 are evolutionary conserved RING finger proteins that can recognize SUMOylated targets through multiple SUMO interaction motifs (SIM) and subsequently trigger Ubiquitination through their RING domains important for dimerization and ubiquitin ligase activity (Hickey et al., 2012; Sriramachandran and Dohmen, 2014). Furthermore it was proposed that SUMOylated telomere bound proteins targeted to the NPC could be de-SUMOylated by the SUMO protease Ulp1 located at the nuclear basket (Zhao et al., 2004), since efficient type II telomere recombination requires Ulp1 localization at the Nuclear Pore (Churikov et al., 2016) (Figure 6 ).
Figure 6: Eroded telomeres are targeted to nuclear pores.
Proteins bound to eroded telomeres are SUMOylated by Siz1/Siz2 and recruited to the Nuclear Pore via the STUbL Slx5/Slx8. SUMOylated proteins are removed possibly through proteasomal degradation (not shown) or through de-SUMOylation by the SUMO protease Ulp1 located at the Nuclear Pore (not shown). These events trigger repair and telomere repeat amplification by type II recombination. Image modified from (Horigome and Gasser, 2016).
Persistent DNA double strand break relocation at the nuclear envelope Similar chromatin dynamics mechanisms have been described for persistent Double Strand Breaks (DSBs) that cannot be repaired by homologous recombination (HR) (Nagai et al., 2008) (Figure 7). It was shown that depending on the cell cycle phase, irreparable DSBs are either recruited to nuclear pores or to the nuclear envelope through interaction with Mps3 (Horigome et al., 2014). In G1 and S phase, proteins bound to irreparable DSBs are poly-SUMOylated by the E3 ligases Siz2 and Mms21 promoting interaction with the STUbL Slx5-Slx8 at nuclear pores (Horigome et al., 2016). In S phase, proteins bound to irreparable DSBs and mono-SUMOylated by Mms21 relocate the persistent DSB to the Mps3 docking site independently of Slx5-Slx8 (Horigome et al., 2016). The authors speculate that the irreparable DSB relocation at the nuclear pore leads to the proteasomal degradation of proteins targeted by the STUbL Slx5-Slx8 (Nagai et al., 2008), an event important to induce alternative repair pathways such us Break Induced Replication (BIR) (Horigome et al., 2016). The clustering or sequestration of irreparable DSBs at Mps3 (Kalocsay et al., 2009; Oza et al., 2009) induced by mono-SUMOylation during S-phase (Horigome et al., 2016;
Horigome et al., 2014) is explained as a cell strategy to prevent ectopic Homologous Recombination.
Figure 7: The recruitment of irreparable DSBs to Nuclear Pores or Mps3 docking sites depends on SUMOylation.
Persistent DSBs are recruited to the nuclear pore in G1 and S phase through poly-SUMOylation of DSB-bound proteins recognized by Slx5-Slx8. This could lead to proteasomal degradation and non-canonical recombination by Break Induced Replication (BIR). In S phase, mono-SUMOylation of DSB-bound proteins results in the recruitment to Mps3 docking sites at the nuclear envelope likely to prevent aberrant Homologous Recombination. Figure from (Horigome and Gasser, 2016).
In contrast to persistent DSBs, repairable DSBs can be repaired by Non Homologous End Joining (NHEJ) when they occur in G1, and Homologous Recombination (HR) when they occur in S and G2 phase. NHEJ is an error prone
repair pathway since it ligates the two DNA extremities without using a homologous template, while HR is an error free pathway since it uses the homologous DNA strand for the repair (Figure 8).
Figure 8: DSB repair by NHEJ in G1 phase or HR in S and G2 phase.
In yeast, Double Strand Breaks are recognized by the MRX complex (Mre11-Rad50-Xrs2). In G1, the Non Homologous end Joining (NHEJ) pathway repairs DSBs thanks to the recruitment of the Ku70-Ku80 complex and the ligation by Dnl4-Lif1-Nei1. In S and G2 phase, DSBs are repaired by Homologous Recombination (HR). The MRX complex and Sae2 enzyme start the 5’!3’ exonucleolytic process. Extensive resection is mediated by the exodeoxyribonuclease 1 (Exo1) and the Dna2-Sgs1 DNA-end processing enzyme. The generated DNA single strands are coated by the Recombination Protein A (RPA) complex; subsequently Rad52 displaces RPA with Rad51 allowing initiation of strand invasion and Homologous Recombination to occur. Figure modified from (Papamichos-Chronakis and Peterson, 2013).
Efficient repair by HR correlates with increased mobility of DSBs in the nucleus especially in diploid cells, in which overall chromatin mobility is enhanced
presumably to favor the search for homologous sequences (Mine-Hattab and Rothstein, 2012).
In haploid cells a single DSB or treatment with low levels of zeocin (DSB-causing drug) do not change genome-wide chromatin dynamics although the damaged locus shows increased intranuclear mobility (Dion et al., 2012). In contrast, prolonged exposure to Zeocin (up to 6h), which causes damage in 60% of the cells, increases overall chromatin dynamics (Herbert et al., 2017). Furthermore, DSBs generated by collapsed replication forks do not show increased chromatin movement, reflecting the absence of need to search for homologous sequences to be repaired, since the template is already present on the sister chromatid (Dion et al., 2012). These observations indicate that chromatin mobility depends on the type of DNA damage and favored repair process.
Finally, it was observed that multiple DSBs cluster together in repair centers characterized by the co-localization with the repair factor Rad52, a situation likely favored by increased chromatin movements (Lisby et al., 2003) (Figure 9).
Interestingly, spontaneous S-phase damage recognized by Rad52 shows reduced mobility and is located within the nucleus, consistent with the mobility of DSBs repaired based on sister chromatid homology (Dion et al., 2013). Notably, when a DSB occurs within the rDNA locus, it has to move outside the nucleolus to form a Rad52 focus and be repaired. If repair occurred within the nucleolus by HR, it would lead to rDNA hyperrecombination and excision of rDNA circles (Torres-Rosell et al., 2007).
Figure 9: Changes in Chromatin dynamics upon DNA damage and formation of Rad52 foci, repair centers for HR.
Upon DNA damage, DSBs cluster in repair centers characterized by co-localization of Rad52, a repair protein important for HR to occur. Rad52 foci always form in the nucleoplasm or nuclear interior, next to the nucleolus, also when the DSB is formed at the rDNA locus (nucleolus in yellow). Image modified from (Lebeaupin, 2015)