Functional Interplay between the 53BP1-Ortholog Rad9 and the Mre11 Complex Regulates Resection, End-Tethering and Repair of a Double-Strand Break

Summary

DNA double strand breaks (DSBs) are among the most deleterious types of damage occurring in the genome, as failure to repair these lesions through either non-homologous-end-joining (NHEJ) or homologous recombination (HR) leads to genetic instability. The 5′ strand of a DSB can be nucleolytically degraded by several nucleases and associated factors, including Mre11, CtIP/Sae2, Exo1 and Dna2 together with Bloom helicase/Sgs1, through a finely regulated process called DSB resection. Once resection is initiated, error-prone NHEJ is prevented. Several findings suggest that DSB resection is a double-edged sword, if not finely regulated, since on one hand it is needed for faithful HR, but on the other it may lead to extensive DNA deletions associated with genome instability. Both in mammals and yeast, 53BP1/Rad9 protein binds near the lesion and counteracts the resection process, limiting the formation of ssDNA. By using S. cerevisiae as a model organism, here we show that Rad9 oligomers block the removal of hypo-active Mre11 protein from a persistent DSB, thus limiting initiation of resection and the recruitment of the recombination factor Rad52, in the absence of Sae2. Altogether, these findings pinpoint a critical role of 53BP1/Rad9 in balancing HR and NHEJ repair events throughout the cell cycle.

Introduction

Similarly to what is seen in higher eukaryotes, in S. cerevisiae the ends of a double-strand DNA break (DSB) are recognized and bound by the Mre11-Rad50-Xrs2 (MRX) complex and the Ku70-Ku80 heterodimer, which compete for end binding. Once the MRX complex, together with CDK1-phosphorylated Sae2 (CtIP in human), initiates resection of the DNA ends, Ku70-Ku80 binding and NHEJ (non-homologous end-joining) are prevented [1], [2], [3], [4]. Subsequent 5′–3′ long-range resection can then occur by one of two pathways: the first utilizes the RecQ helicase Sgs1 (BLM in human), in cooperation with the endonuclease Dna2, and the second utilizes the exonuclease Exo1 [5], [6], [7], [8], [9].

The regulation of DSB end resection is very important to choose the right pathway to repair a DSB and avoid chromosomal rearrangements [10], [11]. Whereas classical NHEJ requires little or no resection, HR (homologous recombination) is characterized by extensive exonucleolytic degradation of one strand. Blocking DNA end resection affects the efficiency and accuracy of how a DSB is repaired. For example, inhibiting resection leads to de novo telomere addition, and eventually loss of a portion of a chromosome [12], [13]. On the other end, extensive DNA end resection could lead to accumulation of unstable DNA intermediates and eventually to the highly error-prone microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA) events, which may cause DNA deletions and translocations [14], [15], [16].

It is now clear that the DNA damage checkpoint response (DDR) plays a central role in regulating DSB end resection. In fact, while resection proceeds, the formation of RPA-coated ssDNA activates the upstream kinase Mec1 (ATR in mammals) and the effector kinase Rad53 (Chk2 in mammals), which in turn phosphorylates and inhibits Exo1 [17]. Interestingly, Exo1 is regulated through a DDR pathway in human cells, too [18], [19].

Moreover, studies both in yeast and mammals showed that Exo1 and other DNA end-processing enzymes are inhibited through a physical structural “barrier” formed by Rad9 oligomers (53BP1 in mammals) bound near a DSB [10]. RAD9 was originally identified as the first checkpoint gene in S. cerevisiae and recognized as an “adaptor” protein, linking the upstream kinase Mec1 to the activation of effector kinases Rad53 and Chk1. Rad9 is recruited to chromatin through three different pathways: i) the constitutive interaction with the histone H3 methylated at the K79 residue by Dot1 [20], [21], [22]; ii) the binding to the histone H2A phosphorylated at the S129 residue by Mec1 [23]; iii) the interaction with Dpb11 [24], [25]. In particular, phospho-H2A mediated Rad9 recruitment spreads many kilobases around a DNA lesion [26]; whereas Dpb11 appears to be more specific at the site of lesion, by binding to a damage-induced phosphorylation in the Ddc1 subunit of the 9-1-1 complex [25], [27], [28]. All of these three pathways cooperate for efficient checkpoint arrest and cell survival after genotoxic treatments throughout the cell cycle. Moreover, Rad9 contains motifs that are necessary for its oligomerization and DNA damage checkpoint signalling [24], [29], [30].

Notably, the Rad9-mediated inhibition of DSB resection is a regulatory function conserved throughout evolution. In fact, 53BP1 facilitates NHEJ at the expense of HR, protecting DNA ends from inappropriate 5' resection, in cooperation with the telomere binding protein RIF1 [31], [32], [33], [34], [35].

Here, we show that in the absence of Sae2, or in presence of mutations affecting Mre11 nuclease activity, Rad9 dimers and/or oligomers, recruited near a DSB mainly by Dpb11 interaction, inhibit the short-range DNA end processing, thereby preventing Mre11 removal from the lesion and limiting Rad52 recruitment by an Sgs1-dependent mechanism. As a consequence, DSB ends cannot be kept efficiently tethered to each other, and repair through an SSA process is prevented. We propose a novel molecular role of Rad9/53BP1 to protect genome integrity from extensive DNA degradation and rearrangements during DSB repair, also suggesting important implications for malignant transformation in mammalian cells.

Results

Deletion of RAD9 gene rescues DSB repair defect in sae2Δ cells through an Sgs1-Dna2 dependent pathway

It is known that deletion of the RAD9 gene in yeast leads to faster DSB resection and repair through an SSA process [36], [37]. To further understand the role of Rad9 in DSB processing and repair, we decided to combine the deletion of RAD9 gene with mutations in genes encoding factors either involved in the short-range (SAE2), or the long-range (EXO1, SGS1) DSB resection [38]. We took advantage of the YMV80 background, in which the galactose-induced expression of the HO nuclease causes a single DSB at a specific site on chromosome III. Repair of this DSB occurs mainly through SSA between flanking homologous leu2 repeats one of which is 25kb from the DSB [39]. We deleted RAD9, EXO1, SGS1 and SAE2 to obtain all viable single, double and triple mutant combinations. Although the sae2Δ sgs1Δ double mutant is a synthetic lethal combination [40], [41], rad9Δ interestingly suppresses sae2Δ sgs1Δ lethality (S1A Fig.). Therefore, it was possible to test the sae2Δ sgs1Δ rad9Δ triple mutant cells. After plating the cells in the presence of galactose to induce one DSB, we found that viability of the sae2Δ and sgs1Δ single mutant and sgs1Δ exo1Δ double mutant was severely reduced (Fig. 1A), as expected [6], [7], [42]. We also found that the deletion of RAD9 gene effectively rescued the viability of the sae2Δ, sgs1Δ and sae2Δ exo1Δ mutant strains following one DSB (Fig. 1A). Interestingly, the viability of the sae2Δ sgs1Δ rad9Δ and exo1Δ sgs1Δ rad9Δ triple mutant cells was very low in the presence of one DSB. Moreover, the HO-induced lethality of the sae2Δ sgs1Δ rad9Δ mutant was not rescued by the expression of the Sgs1-K706A protein variant (S1B Fig.), whose helicase activity is severely reduced [43]. While the failure to repair the DSB in the exo1Δ sgs1Δ rad9Δ triple mutant was expected, since at least one of the Exo1 and Sgs1-dependent pathways is necessary to extensively resect a DSB, the result obtained with the sae2Δ sgs1Δ rad9Δ mutant was surprising. We therefore concluded that an Exo1-independent, Sgs1-dependent pathway is necessary for the viability of sae2Δ cells following a DSB in the absence of RAD9.

Since Sae2 stimulates the activity of the MRX complex in the first step of the DSB end processing [44], we considered the possibility that RAD9 deletion may also rescue an Mre11 nuclease defective mutant or the rad50Δ mutant, in which the MRX complex is disassembled. Interestingly, we found that rad9Δ suppresses the nuclease-defective mre11-D56N mutant [45], through an SGS1-dependent pathway, while it does not rescue rad50Δ mutant, as expected [36] (Fig. 1B). These results suggest that the nuclease activity of the MRX complex is dispensable for the DSB repair in rad9Δ cells; however, the MRX complex must be physically present, likely playing an essential structural role. Indeed, rad50Δ mutation does not rescue sae2Δ cell viability following a DSB (Fig. 1B). Of note, deletion of RAD9 also suppresses the double mutant mre11-D56N sae2Δ, further indicating that Mre11 and Sae2 work together in the same pathway (Fig. 1B).

Importantly, the deletion of RAD9 rescues sae2Δ cell viability through an EXO1-independent, SGS1-dependent pathway also in presence of camptothecin (Fig. 1C), a topoisomerase-aborting agent that causes formation of end-blocked DSBs [46].

To further investigate the findings shown in Fig. 1A at the molecular level, we tested the kinetics of DSB repair by Southern blotting in cells blocked in G2/M cell cycle phase by nocodazole. In agreement with the cell lethality reported in Fig. 1A, we found that the efficiency of the DSB repair is reduced in both the sae2Δ and sgs1Δ single mutants, as previously described [6], [7], [42], and it is severely compromised in sae2Δ sgs1Δ rad9Δ (Figs. 2B and 2C). On the contrary, DSB repair is accelerated and very efficient in the rad9Δ, sae2Δ rad9Δ and sgs1Δ rad9Δ mutants (Figs. 2B and 2C). These results indicate that, in the absence of Rad9, an Sgs1-dependent mechanism is necessary to efficiently repair a DSB in sae2Δ cells.

To test if Sgs1 cooperates with Dna2 to repair a DSB in sae2Δ rad9Δ mutant cells, we took advantage of an auxin-based degradable Dna2 protein variant (Dna2-DEG). This is a common genetic strategy to induce the degradation of a protein by the addition of auxin compound to the cell culture medium [47], and it is particularly useful in the case of an essential gene, such as DNA2. By Southern blotting analysis, we found that the sae2Δ rad9Δ double mutant cells do not repair a DSB in the absence of Dna2 (Fig. 2D and 2E). Therefore, taking all the data in Fig. 2 together, we concluded that the deletion of RAD9 rescues sae2Δ cells through a DSB resection mechanism mediated by the Sgs1-Dna2 pathway.

In addition, we ruled out the possibility that in the absence of Rad9, the DSB can be repaired more efficiently through a strand invasion-based mechanism (such as a break-induced replication process [48]). In fact, we observed faster DSB repair and high viability when we analysed the sae2Δ rad9Δ rad51Δ triple mutant, in which break-induced replication is impaired, but SSA is not inhibited (S2 Fig.).

Rad9 limits an Sgs1- and Sae2- dependent initial step of DSB processing

A critical step to repair a DSB through SSA is 5′ to 3′ resection of the DSB end. Therefore, based on our results in Figs. 1 and 2, we hypothesized that in sae2Δ sgs1Δ rad9Δ triple mutant DSB resection may be affected, as it was shown in the sae2Δ single mutant [6], [7], [42], while it should be faster in sae2Δ rad9Δ double mutant. To test the kinetics of DSB processing we used JKM139 background derivatives, where prolonged expression of HO causes an irreparable DSB at MAT locus, because of the absence of HML and HMR homologous cassettes. Therefore, the analysis of the formation of the 3′ single-stranded (ss) DNA is not biased by a repair process [49]. Using Southern blotting of denatured DNA after restriction enzyme digestion [50], we tested the formation of the 3′ ssDNA filament (as depicted in Fig. 3A), after the induction of one DSB in each sister chromatid, in G2/M-blocked cells.

As expected, we found that the formation of a long 3′ ssDNA tail is slightly delayed in the absence of SAE2, EXO1 and SGS1 genes, and it is severely compromised in the exo1Δ sgs1Δ double mutant [6], [7], [51]. Interestingly, we found more extensive 3′ ssDNA in the absence of Rad9 in all the mutants tested, except the exo1Δ sgs1Δ rad9Δ triple mutant (Figs. 3B, 3C and S3). These results support the model that both the Exo1 and the Sgs1-dependent pathways cooperate to resect a DSB, and rule out the hypothesis that additional nuclease(s) may take over to process a DSB in the absence of Rad9. However, we noticed that in the sae2Δ sgs1Δ rad9Δ triple mutant strain the appearance of ssDNA is slightly delayed compared to wild type and sae2Δ rad9Δ strains (Figs. 3B and 3C). This result may suggest that the initiation of DSB resection is affected in sae2Δ sgs1Δ rad9Δ cells.

To test more precisely DNA processing near a DSB we employed a quantitative PCR-based method [52]. In particular, by this procedure we determined if the RsaI restriction enzyme can cut the DNA at a specific site 150 bp from the HO-cut site, thus indicating whether DSB resection has already passed beyond this site, since, as resection proceeds, the RsaI site becomes single stranded and resistant to digestion, which results in a PCR fragment amplification (see scheme in Fig. 3D). Thus, the rate of PCR fragment amplification, normalized to the efficiency of HO cutting, corresponds to the rate of resection [52]. We also tested with the same procedure another RsaI site 4800 bp from the HO cut site, as a control. Interestingly, we noticed a higher amount of un-resected DNA at 150 bp proximal the DSB site, between 60 and 180 minutes after the cut in nocodazole blocked sae2Δ and sae2Δ sgs1Δ rad9Δ triple mutant cells with respect to the wild type and sae2Δ rad9Δ mutant (Fig. 3E). However, at later time points resection has efficiently passed beyond the RsaI site 4800 bp far from the HO cut site (Fig. 3F), not only in the wild type and sae2Δ rad9Δ cells, but also in the sae2Δ sgs1Δ rad9Δ triple mutant cells, according to the visualization of the 3′ ssDNA formation by denaturing Southern blotting (Figs. 3B and 3C).

These studies revealed one striking unexpected result: although sae2Δ sgs1Δ rad9Δ triple mutant cells resect a DSB and expose an extended 3′ ssDNA (Figs. 3B, 3E and 3F), they are severely compromised in DSB repair through SSA (Figs. 2B and 2C), suggesting that the long-range resection is not the limiting step to repair a DSB in these cells, rather the defect is different from simply creating enough ssDNA to allow SSA to take place. Therefore, we hypothesize that an Sgs1-dependent mechanism contributes to efficiently initiate DSB processing in the absence of both Rad9 and Sae2, and the kinetics of the initial step of resection would become somehow critical to complete the subsequent steps of the SSA repair.

We then investigated whether the faster DSB end processing that we observed in sae2Δ rad9Δ cells would be associated with reduced NHEJ events, which are significantly elevated in the sae2Δ cells [53]. To this aim, we treated cells of JKM139 strains with nocodazole to block cell cycle in G2/M phase and we added galactose to induce one persistent DSB in each sister chromatid. Cells were kept in nocodazole for 2 hours to avoid potential interference caused by cell cycle transition, before plating in the presence of galactose. In this condition, the continued expression of HO leads to a recurrent cut of the MAT locus and precludes precise religation, until the sequence of the HO site is corrupted by deletion/addition of few bases and the ends are joined by imprecise NHEJ [54]. This is a relatively inefficient process in yeast, with a frequency of about 1-3×10⁻³ in wild type cells [54]. We found that the frequency of imprecise NHEJ events is increased in sae2Δ cells, in agreement with previous finding [53], while it is slightly reduced in the absence of Rad9. Interestingly, deletion of RAD9 reduces NHEJ events to wild type value in sae2Δ cells (Fig. 3G).

These results suggest that Rad9 plays a critical role to balance NHEJ and HR events in G2/M phase, likely acting at an early step of DSB processing, leading to increased NHEJ events in the absence of Sae2.

Rad9 limits Mre11 removal from a DSB, affecting Rad52 binding and DSB end-tethering in sae2Δ cells

The delay in DSB resection in sae2Δ cells has been correlated with a prolonged Mre11 binding at the DSB site [42], [55]. More recently, it was also shown that an Sgs1-dependent process can contribute to remove Mre11 from a DSB in sae2Δ cells, promoting DSB resection and repair through homologous recombination [56]. Therefore, we decided to investigate Mre11 binding near a DSB by a chromatin immunoprecipitation-after-crosslinking-protocol (ChIP), followed by quantitative PCR (qPCR), with primers specific for the DSB site. Contrary to wild type, rad9Δ or sgs1Δ cells, we found greater and persistent levels of Mre11 bound near DSB ends in sae2Δ cells (Fig. 4A), supporting previous analysis of the Mre11 foci by microscopy [51], [56], and by ChIP [55]. Importantly, we found a decrease in fold enrichment of Mre11 binding to the DSB site in sae2Δ rad9Δ cells, but not in the sae2Δ sgs1Δ rad9Δ triple mutant cells (Fig. 4B). These results suggest that the deletion of RAD9 gene promotes an Sgs1-dependent process to remove Mre11 from DSB ends in the absence of Sae2, supporting and expanding recent findings [56], and it may explain the high efficiency of SSA repair and viability of the sae2Δ rad9Δ that we showed in Figs. 1 and 2. Moreover, the prolonged binding of Mre11 near the DSB further supports previous results in Fig. 3, showing that short-range resection in the sae2Δ and sae2Δ sgs1Δ rad9Δ triple mutant cells is delayed.

Since it is known that Mre11 persistence at a DSB limits the recruitment of Rad52 [4], [57], which is necessary to establish DNA end-tethering and HR pathways [58], [59], we investigated by immunofluorescence Rad52 loading onto one DSB in all the mutants described. We found that deletion of RAD9 totally restores Rad52 binding in sae2Δ cells through an Sgs1-dependent mechanism (Fig. 4C). These results correlate with the analysis of Mre11 binding in these mutants (Fig. 4B), and suggest that the limiting step to efficiently complete an SSA process in nocodazole-blocked sae2Δ and sae2Δ sgs1Δ rad9Δ cells is not the delay in DSB resection per se (Figs. 3B and 3C), but rather the reduced binding of Rad52.

Rad52 is a critical factor to maintain DSB ends tethered to each other, which was suggested to be a relevant event in HR [42], [58], [59], [60], [61]. As we showed that the deletion of RAD9 allows Rad52 binding in sae2Δ cells (Fig. 4C), we investigated whether it may also contribute to rescue DSB end-tethering defect in these cells. To this end, we took advantage of a specific yeast background in which the DNA proximal to the irreparable HO break could be visualized by binding of a LacI-GFP (green fluorescent protein) fusion protein to multiple repeats of the LacI repressor binding site, LacO. These arrays are integrated at a distance of 50 kb on either side of the HO cleavage site on chromosome VII [58]. Cultures of the original wild type and isogenic sae2Δ, sae2Δ rad9Δ and sae2Δ sgs1Δ rad9Δ derivative strains were arrested in mitosis and kept blocked by nocodazole treatment during break induction by galactose addition. After 2 hours to ensure HO cut formation, we observed two LacI-GFP spots in only 12.5%±2.1% of the wild type cells, and 11.0%±3.1% in sae2Δ rad9Δ mutant cells, thus indicating their ability to hold the broken DNA ends together. In contrast, 42.3%±3.8% of sae2Δ and 42.5%±4.8% of sae2Δ sgs1Δ rad9Δ cells showed two LacI-GFP spots, indicating a failure in DSB end-tethering (Fig. 4D, and see also [42], [62]).

Therefore, we conclude that the deletion of RAD9 rescues both the Rad52 binding and DSB end-tethering in sae2Δ cells, contributing to efficiently repair a DSB through an SSA process that requires the resection of 25 kb of DNA between the repeats (Fig. 2A).

Rad9 oligomers limit sae2Δ cells viability following a DSB mainly through the interaction with Dpb11

It was previously suggested that Rad9 limits DSB resection acting as a physical barrier toward the actions of nucleases, through a function distinct from its role in DNA damage checkpoint signalling [10]. Therefore, we sought to address if a checkpoint-independent function of Rad9 was involved to limit sae2Δ cells viability following one DSB. To this aim, we tested the chk1Δ rad53-K227A double mutant in the YMV80 background, in which the Rad53 kinase activity is dead and both the two checkpoint-signaling pathways acting downstream Rad9 are abrogated. By plating the cells in the presence of galactose to induce one HO cut, we found that the viability of the sae2Δ chk1Δ rad53-K227A triple mutant cells is reduced, similarly to sae2Δ cells (Fig. 5A). This result indicates that signaling through Rad53 and/or Chk1 is not involved into the mechanism by which Rad9 limits SSA repair in sae2Δ cells.

In order to further understand how Rad9 inhibits SSA repair in sae2Δ cells, we then investigated specific mutations that affect Rad9 binding to a DSB. It is known that Rad9 constitutively binds chromatin through the interaction between its TUDOR domain and the histone H3 methylated at the K79 by Dot1 [20], [21], [22]. In addition, Rad9 binds chromatin around a DSB site through the interaction of its BRCT domain with the histone H2A phosphorylated at the S129 (γ-H2AX) by upstream kinase Mec1 and Tel1 [23]. Further, Rad9 is recruited near a DNA lesion through the interaction with Dpb11 protein. In particular, Dpb11 binds the CDK1-dependent phosphorylated S462 and T474 Rad9 residues, reinforcing the Rad9 binding to damaged DNA and promoting Rad9 phosphorylation by Mec1 [25].

To test the contribution of the different pathways that mediate Rad9 binding to chromatin, we analysed the viability in the presence of HO-induced DSB of specific mutations that abrogate each of them in the YMV80 background. The deletion of DOT1 gene eliminates the H3K79 methyl transferase Dot1 protein, and greatly reduces the constitutive binding of Rad9 to chromatin [21], [24]. As expected [36], deletion of DOT1 leads to a faster long-range DSB resection in sae2Δ cells (S4A and S4B Figs.). However, by the qPCR-based method, we found that the initial short-range resection is still delayed in these double mutant cells (S4C Fig.), suggesting that the Dot1-dependent resection barrier may have a role only at distal region from the cut site. Indeed, by plating the YMV80 derivative cells in the presence of galactose to induce one DSB, we found that deletion of DOT1 gene does not rescue sae2Δ lethality (Fig. 5A). Further, we deleted SAE2 gene in a strain that expresses the H2A-S129A histone variant, which is not phosphorylatable by Mec1 and Tel1 kinases and leads to a faster DSB resection [63]. We also deleted SAE2 gene in a strain that expresses the Rad9-S462A-T474A (hereafter we refer to rad9-S462A-T474A as rad9-2A) protein variant, which does not interact with Dpb11 [25]. Interestingly, both the failure to phosphorylate the H2A-S129 site and the rad9-2A mutation increase the viability of sae2Δ cells after one DSB, with the major contribution done by the mutation that abrogates the Rad9-Dpb11 interaction (Fig. 5A). Taking all these genetic results together, we concluded that the recruitment of Rad9 near the DSB site, mediated by its interaction with Dpb11 and partially with γ-H2AX, limits sae2Δ cells viability when a DSB must be repaired by SSA.

Consistently with our genetic evidence, we found an increased binding of Rad9 close to an irreparable DSB in sae2Δ cells by ChIP analysis (Fig. 5B), which correlates with the increased binding of Mre11 (Figs. 4A and 4B). Of note, the Rad9-2A protein variant does not bind near a break (Fig. 5B), supporting the viability data of the sae2Δ rad9-2A double mutant cells following one DSB (Fig. 5A). Moreover, Rad9 binding close to the break is only partially dependent on γ-H2AX and not by Dot1 (S5 Fig.), in agreement with cell viability of the sae2Δ h2a-S129A and sae2Δ dot1Δ double mutants (Fig. 5A).

Then we tested if the capability of Rad9 to form oligomers at the DNA damage site [29], [30], [64] was involved in inhibiting sae2Δ cells viability following a DSB. To this aim, we introduced a plasmid vector that expresses either the rad9-7xA allele or the RAD9 gene as a control, by transformation into rad9Δ and sae2Δ rad9Δ YMV80 derivatives. The Rad9-7xA protein variant cannot be phosphorylated at critical sites by upstream Mec1 and Tel1 kinases (see also Fig. 5C), and is unable to oligomerize [29], [64]. After plating cells in the presence of galactose to induce one DSB, we found that the expression of the Rad9-7xA protein variant rescues the lethality of sae2Δ cells, contrary to the wild type Rad9 (Fig. 5D). This result suggests that the oligomerization of Rad9 molecules is implicated in limiting SSA repair in sae2Δ cells. To further support this conclusion, we took advantage of the rad9-ΔBRCT-FKBP chimeric allele, which leads to the production of a truncated variant of Rad9 protein, in which the C-terminal BRCT domains are replaced with a FKBP tag [24]. It was shown that the Rad9-ΔBRCT-FKBP protein variant, which cannot form oligomers due to the absence of the BRCT domains, can dimerize in the presence of the small inducing molecule AP20187, binds chromatin and partially transduces the checkpoint signal (S6B Fig. and see also [24]). Consistent with our hypothesis, we found that the rad9-ΔBRCT-FKBP mutation does not rescue sae2Δ lethality in the presence of AP20187, while the viability in the sae2Δ rad9-ΔBRCT-FKBP double mutant cells is almost identical to the wild type value (Fig. 5E), further suggesting that the dimerization/oligomerization of Rad9 affects SSA repair.

Discussion

It is now clear that DSB processing is a finely regulated process, which acts at the crossroad between HR and NHEJ recombination pathways. Indeed, as soon as a DSB is resected, homologous recombination pathways can be used to repair the break in lieu of NHEJ, with important implications for chromosome rearrangements and genome integrity.

Similarly to what seen in higher eukaryotes, three distinct nucleases cooperate to resect a DSB in S. cerevisiae. According to a model recently proposed for meiotic DSBs [65], Mre11, activated by Sae2 [44], introduces a nick near a DSB, triggering a bidirectional nucleolytic degradation of the 5′ strand: Exo1 and Dna2-Sgs1 resect the DNA in the 5′-to-3′ direction from the nick, while the Mre11 complex resects the DNA in the 3′-to-5′ direction toward the DSB ends. In G2/M blocked cells, it appears that the Exo1 and Dna2-Sgs1 pathways cannot actively resect a DSB starting from its ends, which are occupied by Ku70-Ku80 complex [1]. Indeed, it was suggested that the Mre11 activity might contribute to the removal of Ku complex, clearing the ends [2], [3], [11], [65], [66]. Importantly, in the absence of a functional Sae2, the Mre11-dependent DSB processing is compromised, and Ku-dependent NHEJ events and translocations increased [62]. In addition, Mre11 and Rad52 binding are, respectively, increased and reduced in sae2Δ cells (Fig. 4, and see [4], [57]), which are severely defective in repairing a DSB through SSA (Fig. 2, and see also [6], [42]). Moreover, sae2Δ cells cannot keep the DSB ends tethered, which was shown to be relevant for DSB repair (Fig. 4, and see [42], [58], [60]). Here, we show that the deletion of the RAD9 gene suppresses all these phenotypes of sae2Δ cells. Indeed, we found that deletion of RAD9 leads to a faster 5′–3′ resection both through the Exo1 and Dna2-Sgs1 pathways, but the Dna2-Sgs1 pathway becomes essential, in the absence of Sae2, to efficiently initiate DSB processing and repair through an SSA process that requires 25 kb DNA resection (Figs. 2 and 3). We also found elevated levels of Mre11 bound near an HO-induced break both in sae2Δ and sae2Δ sgs1Δ rad9Δ mutants, accordingly with a defect in Rad52 binding and DNA end-tethering (Fig. 4). The requirement of DSB end-tethering for SSA repair has never been explored before, however it is relevant to underline that Rad52 is important for end-tethering [58], and also our results indicate that a defect in end-tethering is linked with a failure to accomplish SSA repair. Further investigation will be required to fully understand the interplay between SSA and end-tethering. Interestingly, recent findings underlined a role of exonuclease processing of a DSB in maintaining broken chromosome ends in close proximity [61].

Taken all these findings together, we suggest that the prolonged binding of Mre11 near the break site may represent the critical barrier to efficiently initiate DSB resection, load Rad52 and establish end-tethering in the absence of Sae2, and it can be by-passed by a resection-based mechanism mediated by Sgs1-Dna2 in the absence of Rad9.

A similar role to remove Mre11 from a DSB site in sae2Δ cells was recently shown for Sgs1, in the absence of Ku70-Ku80 complex [56]. Indeed, deletion of KU70 suppresses sae2Δ cells sensitivity to low doses of CPT and other DSB inducing agents [1], [3]. Surprisingly, we did not see a rescue of sae2Δ cells lethality by deleting KU70 after a DSB that can be repaired through an SSA process between two homologous leu2 repeats 25kb far from each other, although deletion of RAD9 suppresses the sae2Δ ku70Δ double mutant (S7 Fig.). One possibility is that Rad9, bound near a DSB site, may limit the Sgs1-Dna2 activity starting from the break ends, leading to prolonged Mre11 binding. This might occur in cooperation with Ku complex, bound to the DSB ends, or rather it might represent a second distinct mechanism to limit DSB ends resection and DNA end-tethering. Alternatively, or in addition, Ku and Rad9 may limit DSB processing in different cell cycle phases. Indeed, the Ku complex acts on a DSB mainly in G1, while Rad9 acts predominantly in G2/M phase [36], [67], [68].

Genetic and biochemical evidence in Fig. 5 suggest that Rad9 protein dimerization and/or oligomerization, together with Rad9 interactions with Dpb11 and partially with γ-H2AX, are important to limit short-range resection and repair in sae2Δ cells. Indeed, Dpb11 is recruited on to the DNA lesion through the interaction with the 9-1-1 complex [28], and both the 9-1-1 complex and Dpb11 are recruited rapidly near a DSB site [69], likely at the ssDNA-dsDNA junction [70]. It is possible that the interactions with γ-H2AX, as well as with the histone H3 methylated at Lys79 by Dot1, become more important to recruit Rad9 in a distal region from the DSB site, contributing to slow down the long-range resection, which is not the limiting step in sae2Δ cells. This hypothesis is supported by the fact that DNA damage sensitivity of fun30Δ cells, that resect slower a DSB because of their inefficient Rad9 removal from chromatin flanking a DSB [37], is partially rescued in the absence of γ-H2AX or Dot1 [37], [63]. Of importance, deletion of DOT1 gene does not rescue sae2Δ cells (Fig. 5A). Notably, although Rad9 binding close to the break is not particularly elevated in wild type cells, it is enriched in sae2Δ cells (Fig. 5C). Consistent with our genetic evidence, Rad9 binding close to DNA ends depends on Dpb11, partially on the histone γ-H2AX, but not on the histone H3 methylated at Lys79 by Dot1 (Figs. 5B and S5). Possibly, these data are in agreement with the low amount of modified histones detected in chromatin within 1–2 kb of the break [22], [26], [71], [72], [73].

Overall, our genetic and molecular results suggest a model shown in Fig. 6, in which Rad9, in addition to its known role in inhibiting long-range resection, may affect the initial short-range processing of an HO-induced DSB. In fact, Rad9, once recruited close to a DSB end in G2 phase mainly through the interaction with Dpb11, limits the Sgs1 dependent resection starting from DNA ends, whenever Mre11 is blocked near the DNA ends. In the future it will be interesting to investigate whether Rad9 plays a similar role in limiting rapid and coincident resection of dirty radiation-induced DSBs, in cells lacking Sae2 and/or Mre11 [74].

We believe that our findings might have important implications for understanding how the genome stability is preserved, especially in higher eukaryotes, whose genomes are enriched of repeats and SSA events can be particularly frequent. In fact, it becomes clear that too-efficient DSB resection can lead to an excessive initiation of homologous recombination and accumulation of toxic DNA intermediates and rearrangements between repeats [16]. Moreover, DSB resection may lead to highly error-prone alternative ends joining (A-EJ) and MMEJ events [14], [16]. In this view, our results in yeast might help to understand recent finding in human cells at the molecular level, showing a role for 53BP1 in protecting from BLM and CtIP-Mre11 dependent A-EJ events and genome rearrangements [75].

Furthermore, our findings suggest that the functional interplay between 53BP1/Rad9 and Mre11 may also have a physiological relevance to protect from error-prone imprecise NHEJ events in genomic regions containing no repeats. It is also worth mentioning that the inactivation of 53BP1 was shown to potentiate homologous recombination and increase DNA damage tolerance of cancer-prone BRCA1 -/- cells [32], [76], [77], [78], with severe implications for therapeutic treatments.

In conclusion, we show novel insights on the structural barrier induced by Rad9, together with Dpb11 and γ-H2AX, to limit DSB processing and repair. The Sgs1-Dna2 pathway becomes essential to efficiently remove hypo-active Mre11 from a DSB site, in the absence of Sae2 and Rad9, triggering DSB resection and repair. The efficient removal of Mre11 from the DSB site is essential not only to switch to the more processive long-range resection, but also to allow an efficient recruitment of the recombination factor Rad52. This allows the maintenance of DSB end-tethering, which is an important prerequisite to complete repair, especially for those lesions that require extensive resection. These events increase in the absence of Rad9 and might contribute to accumulation of toxic HR events, leading to genome rearrangements and genetic instability.

Materials and Methods

Yeast strains, media and growth conditions

All the strains listed in S1 Table are derivative of JKM139, YMV80 and yJK40.6. To construct strains standard genetic procedures of transformation and tetrad analysis were followed. Deletions and tag fusions were generated by the one-step PCR system [79]. For the indicated experiments, cells were grown in YP medium enriched with 2% glucose (YEP+glu), raffinose 3% (YEP+raf) or raffinose 3% and galactose 2% (YEP+raf+gal). All the synchronization experiments were performed at 28°C.

Measurement of DSB resection at MAT locus

DSB end resection in JKM139 derivative strains was analyzed on alkaline agarose gels using a single-stranded RNA probe as described previously [36], [50].

SDS-PAGE and western blot

TCA protein extract was prepared [80] and separated by SDS-PAGE. Western blotting was performed with anti-Rad53 (EL7), anti-HA (12CA5), anti-Rad9 (generously provided by N. F. Lowndes), and anti-actin using standard techniques.

Analysis of SSA repair

Repair of an HO-induced DSB in YMV80 background was analyzed by a Southern blotting procedure described previously [39].

Cell viability assay

YMV80 derivative strains were inoculated in YEP+raf, grown O/N at 28°C. The following day, cells were normalized and plated on YEP+raf and YEP+raf+gal. Plates were incubated at 28°C for three days. Viability results were obtained from the ratio between number of colonies on YEP+raf+gal and YEP+raf. Standard deviation was calculated on three independent experiments.

Non homologous end joining assay

JKM139 derivative strains were inoculated in YEP+raf, grown O/N at 28°C. The following day, after cell cycle block in G2/M by nocodazole, 2% galactose was added to one part of the culture to induce HO cut. After 2 hours of HO induction, cells were normalized and plated on YEP+raf and YEP+raf+gal. Plates were incubated at 28°C for three days. Viability results were obtained from the ratio between number of colonies on YEP+raf+gal and YEP+raf. Standard deviation was calculated on three independent experiments.

ChIP analysis

ChIP analysis was performed as described previously [69]. Input and immunoprecipitated DNA were analysed by quantitative PCR using a Biorad MyIQ2 system or a Biorad CFX connect. The oligonucleotides used are listed in S2Table. Data are presented as fold enrichment at the HO cut site (0.15 or 4.8 kb from the DSB) over that at the PRE1 locus on chromosome V, then normalized to the corresponding input sample. The obtained fold enrichment values were normalized to the fold enrichment of the t₀ sample. Standard mean error (SEM) was calculated on three independent experiments.

Quantitative analysis of DSB end resection by real time PCR

Quantitative PCR (qPCR) analysis of DSB resection was performed accordingly to [52]. The oligonucleotides used are listed in S2 Table. The DNA was digested with the RsaI restriction enzime (NEB) that cuts inside the amplicons at 0.15 kb and 4.8 kb from the DSB, but not in the PRE1 control region on chromosome V. qPCR was performed on both digested and undigested templates using StoS Quantitative Master Mix 2X SYBR Green (Genespin) with the Biorad MyIQ2 PCR system. The ssDNA percentage over total DNA was calculated using the following formula: % ssDNA  =  {100/[(1+2^ΔCt)/2]}/f, in which ΔCt values are the difference in average cycles between digested template and undigested template of a given time point and f is the HO cut efficiency measured by Southern blot analysis.

DSB end-tethering experiment

Cells of strains derivative from yJK40.6 background were grown in YEP+raf and blocked 3 hours in G2 with nocodazole. 160 µM CuSO₄ was added one hour before inducing HO cut with galactose, accordingly to [58]. Samples taken at the indicated time were analysed with a fluorescence microscope. Cells with 2 LacI-GFP foci separated by more than 0.5 µm were considered defective in DSB end-tethering.

Supporting Information

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