Rad9/53BP1 promotes crossover recombination DNA repair by limiting the Sgs1 and Mph1 helicases

A DNA double strand break (DSB) is primed for homologous recombination (HR) repair through the nucleolytic processing (resection) of its ends, leading to the formation of a 3′ single-stranded DNA (ssDNA). Generation of the ssDNA is accompanied by the loading of several repair factors, including the ssDNA binding factor RPA and the recombinase Rad51. Then, depending upon the availability and location of a homologous sequence, different types of HR mechanisms can occur. Inefficient or slow HR repair results in the activation of the DNA damage checkpoint (DDC)1. In budding yeast, the 53BP1 ortholog Rad9 acts as a scaffold, mediating signal from upstream kinases Mec1 and Tel1 (ATR and ATM in human) to downstream effectors kinases Rad53 and Chk1 (CHK2 and CHK1 in human). In addition to its role in DDC, Rad9 limits DSB resection 2. Remarkably, this function is conserved in 53BP1, also being implicated in cancer biology in human cells 3,4. Here we show that Rad9 limits the recruitment of the helicases Sgs1 and Mph1 on to a DSB, promoting Rad51-dependent recombination with long track DNA conversions, crossovers and break-induced replication (BIR). This regulation couples the DDC with the choice and effectiveness of HR sub-pathways, and might be critical to limit genome instability with implication for cancer research.


Main
Rad9 is the 53BP1 ortholog in S. cerevisiae (budding yeast). It plays fundamental role in DNA damage signalling, mediating phosphorylation from upstream kinases Mec1/ATR and Tel1/ATM. In addition to this function, we described a DDC-independent role of Rad9 in the regulation of DSB resection, limiting repair through single strand annealing (SSA). Indeed, the Rad9 binding to a DSB reduces the Exo1 and Dna2-Sgs1 recruitment, affecting the long-range resection 2,5,6 . However, it is not understood how the formation of the 3′-end filament is coupled with the selection of appropriate HR sub-pathway, and whether Rad9 might have a role to determine that choice.
To investigate further the role of Rad9 in DSB repair, we performed a genetic assay with a diploid system, which allows to study the repair of DSB through gene conversion (GC) and break-induced replication (BIR), after the induction of a DSB by I-SceI in chromosome XV 7 . Importantly, this system not only measures frequencies of crossover (CO) and noncrossover (NCO) in DSB repair, but also distinguishes between short and long tract gene conversion events (Fig. 1a).
Upon I-SceI induction, in the absence of RAD9 we observed a striking increase in the percentage of short track GC (white colonies in the assay) (Fig. 1b). By screening colonies for specific markers, we also found that rad9Δ cells have reduced frequency of both CO and BIR events (Fig. 1c, and Extended data Table   1). Based on these results, we hypothesized that Rad9 might control strand invasion-mediated mechanisms to repair a DSB, in addition to its function in limiting SSA.
To study more precisely the role of Rad9 in the BIR process, we took advantage of an haploid genetic system engineered to test only this HR subpathway 8 .
Briefly, one DSB is induced in chromosome V by the endonuclease HO and it is repaired by BIR thanks to a donor sequence on chromosome XI (Fig. 1d).
Interestingly, analysing the DSB repair by Southern blotting, in exponentially growing or nocodazole-arrested cells (Fig. 1e, f), we found that RAD9 deletion severely affected BIR, regardless of the cell cycle stage. Considering that DDC signalling to Pif1 helicase contributes to BIR 9 , we speculated that this would be the reason of the rad9Δ cells defect in the assay. However, after plating the cells in galactose to induce HO, we surprisingly observed higher cell lethality in rad9Δ cells with respect to rad53-K227A chk1Δ cells, that are defective in DDC signalling downstream from Rad9, and pif1Δ cells (Fig. 1g). Therefore, cells lacking RAD9 would be defective in BIR for a reason other than deficient signalling to Pif1.
Then, we tested critical steps in Rad51-dependent HR in rad9Δ cells. First, by chromatin immunoprecipitation (ChIP), we found that the recombination factors Rpa1, Rad52 and Rad51 were efficiently recruited at the cut site in rad9Δ cells ( Fig. 2a, b, c, and Extended data Fig. 1). However, their binding near to the break ends was significantly higher in the absence of RAD9, especially at later time points. Since the amount of ssDNA close to the break site in wild type and rad9Δ cells was similar (Extended data Fig. 2), the increased recruitment of Rpa1, Rad52 and Rad51 on the DSB was unlikely due to higher amount of available substrate in rad9Δ cells. Instead, we hypothesized that the loading and oligomerization of Rad9 protein on the DSB might physically dampen the recruitment of the recombination factors, similarly to its role in limiting nucleases for DSB resection 2,5,6 . To test this hypothesis, we expressed from a plasmid the wild type Rad9 or the two protein variants Rad9-2Ala and Rad9-7xA, both reducing the Rad9 binding and oligomerization on the DSB 5,10 , in rad9Δ cells. We found that both the Rad9 variants, contrary to the wild type form, did not completely rescue the lethality of rad9Δ cells in the BIR assay (Extended data Fig. 3), supporting the idea that Rad9 might affect BIR though a physical role at the DSB site.
Despite the Rad51 binding at the DSB was increased (Fig. 2c), we found that it was not enriched at the donor site in rad9Δ, after DSB induction in G2/M blocked cells (Fig. 2d). This result indicates that cells lacking RAD9 cannot form a stable Displacement-loop (D-loop) structure and synapsis between DSB and the donor template, providing molecular evidence of the failure in DSB repair by BIR.
Consistent with these data, RAD9 deletion impaired the D-loop extension, measured through a PCR-based assay to a greater extent than rad53-K227A chk1Δ and pif1Δ mutations (Fig. 3a, b, c). These results suggest that Rad9 promotes strand invasion and D-loop extension in BIR, through a mechanism independent on the Chk1 and Rad53 signalling. Of note, previous works have shown that the annealing between the two DNA strands in D-loop formation can either be promoted by a Rad51-dependent process or rejected by the Sgs1-Top3-Rmi1 complex and Mph1 11-15 . Strikingly, after deletion of the genes coding for the two helicases Sgs1 and Mph1 in rad9Δ cells, both viability and primer extension analyses showed a rescue to the level found in pif1Δ cells (Fig. 3d, e, f).
Moreover, by ChIP analysis, we also found higher binding of Sgs1 and Mph1 on to a persistent DSB in rad9Δ cells (Fig. 3g, h). As Sgs1 interacts with Rpa1 and Rad51 16,17 , while Mph1 interacts with Rpa1 and Rad52 18,19 , we are tantalized to suggest that the hyper-loading of Sgs1 and Mph1 at the DSB may be related to the increased recruitment of Rad51, Rad52 and Rpa1 in rad9Δ cells (Fig. 2a, b, c, and Extended Fig. 1). In particular, it has been recently shown that Sgs1 interacts with Rad51 and the sgs1-F1192D (sgs1-FD) mutation abolishes this interaction 17 .
We found that the sgs1-FD partially rescued the viability of cells lacking RAD9 in the BIR assay (rad9Δ= 1.29 % ± 0.32; sgs1-FD rad9Δ= 2.67 % ± 0.03; mph1Δ sgs1-FD rad9Δ= 8.25 % ± 0.11) (Fig. 3d, i), similarly to the deletion of SGS1 (sgs1Δ rad9Δ= 5.73 % ± 0.83; mph1Δ sgs1Δ rad9Δ= 11.87 % ± 1.78) (Fig. 3d). This result suggests that lowering the interaction between Sgs1 and Rad51 might reduce strand rejection, favouring BIR in rad9Δ cells. As because the interplay between Rad9 and Sgs1 regulates also DSB resection 5,6 , we measured the kinetic of ssDNA formation at different positions from the break in the sgs1-FD mutant. We found that this allele did not alter resection of an irreparable DSB, which still remained faster in the sgs1-FD rad9Δ double mutant cells (Extended data Fig. 2). These results, together with the viability of the sgs1-FD rad9Δ cells in the BIR assay ( Fig. 3i), suggest that the kinetic of the DSB resection is not responsible per se of the severe BIR defect of the rad9Δ cells.
Because of the reduced levels of CO and long track gene conversion in rad9Δ cells (Fig 1a, b, c), we investigated the role of Rad9 in DSB repair by using an ectopic gene conversion (eGC) system 11 , which is coupled to DDC activation 20 . This assay allows us to detect both the CO and NCO repair products by Southern blotting (Fig 4a). Of note, deletion of SGS1 and MPH1 greatly increase CO 11,12 .
Significantly, we found that rad9Δ cells had reduced repair product through both NCO and CO, with a major effect on the latter, when the DSB was induced in cells blocked in G2/M phase (Fig. 4b, c). Once again, by deleting SGS1 and MPH1 we observed a partial rescue of the DSB repair defect in rad9Δ cells, specifically for the less frequent class of repair associated with CO (Fig. 4b, c).
Overall, this work provides experimental evidence of an unprecedented role of Rad9 in DSB repair, controlling the fate of 3′ Rad51 filament in HR. Interestingly, limiting the Sgs1 helicase, Rad9 antagonizes the formation of extended 3′ ssDNA for recombination while, limiting both Sgs1 and Mph1, reduces the disassembling of D-loop structure once repair DNA synthesis is commenced (Fig.   4d). This reduces SSA and favours DSB repair through HR sub-pathways that require stable D-loop, such as long tract GC, eGC and BIR, also increasing the frequency of CO outcome. Accordingly, RAD9 deletion limits sister chromatid exchanges and promotes Rad1/XPF-dependent translocations, likely through SSA 21,22 . Moreover, in line with our model, eliminating the SGS1 gene in rad9Δ cells causes dramatic levels of translocations between homeologous sequences and complex chromosomal rearrangements 23,24 .
In conclusion, we suggest that the Rad9 recruitment at a DSB guarantees that the DNA break is engaged into complex and slow HR sub-pathways only when cell division is restrained by the DDC activation. This reduces the risk of premature segregation of chromosomes with DNA linkages caused by unresolved HR intermediates, which leads to the formation of anaphase bridges and deleterious chromosome rearrangements 25,26 . As such, this regulation might be critical to limit genetic instability, a hallmark of cancer cells. Indeed, 53BP1-depleted cancer cells accumulate ultrafine anaphase bridges, suggesting that this mechanism might be evolutionary conserved also in higher eukaryotes 27 .

Methods
Yeast strains, Media and Growth conditions. All the strains listed in Extended data Table 2 are derivative of JKM179, JRL92, tGI354 and W303. To construct strains standard genetic procedures were followed 28 . Deletions and tag fusions were generated by the one-step PCR system. The sgs1-F1192D was obtained using a Cas9 mediated gene targeting system 29 . For the indicated experiments, cells were grown in YEP medium enriched with 2% glucose (YEP+glu), 3% raffinose (YEP+raf) or 3% raffinose and 2% galactose (YEP+raf+gal). Unless specified all the experiments were performed at 28 °C. To block cells in G2/M, 20 μg/ml nocodazole was added to the cell culture.
Cell viability assay. JRL92 derivative strains were inoculated in YEP+raf, grown O/N at 28°C. The following day, cells were normalized and plated on YEP+glu and YEP+gal. Plates were incubated at 28 °C for three days. Viability results were obtained from the ratio between number of colonies on YEP+gal and YEP+glu.
Standard error of the mean (SEM) was calculated on two independent experiments.

Southern blot analysis.
Purified genomic DNA was digested with the appropriate restriction enzyme/s, probed with a specific 32 P-labeled probe and scanned with a Typhoon Imager (GE healthcare). In JRL92 a CAN1 fragment was used as a probe, the % of BIR repair has been calculated using the donor band as a loading control. Repair in tGI354 background was analyzed as described previously 20 . Densitometric quantification of the bands intensity was performed using the ImageJ software. The SEM was calculated on three independent experiments.
ChIP analysis. ChIP analysis was performed as described previously 5 . Input and immunoprecipitated DNA were analysed by quantitative PCR, using a Bio-Rad CFX connect, or droplet digital PCR (ddPCR), using a Bio-Rad QX200 droplet reader. The oligonucleotides used are listed in Extended data Table 3. Data are presented as fold enrichment at the HO cut site (at the indicated distance from the DSB) over that at the KCC4 locus on the left arm of chromosome III or CAN1 locus on chromosome V. Then normalized to the corresponding input sample.
SEM was calculated on two independent experiments.

D-loop extension analysis.
To measure the DNA synthesis after D-loop formation during BIR we adopted a strategy that was described previously 8 . In our experiments, the genomic DNA at 0h and 24h after DSB formation was amplified by PCR with oligonucleotides on the CAN1 locus and, as a control, on TLC1 locus (Expanded data Table 3). See a scheme in Fig. 2b.     Fig. 1, 2, 3, 4 and Expanded data Fig. 1, 2, 3. Fig 1b, c, and contributed to Fig. 2 and Fig. 3. Fig 2c and Fig. 3g, h, and Expanded data Fig. 1a.

S.L. contributed to the ChIP analyses in
A.P. supervised and coordinated all aspects of the work.