The suppression of alt-EJ in quiescent G0-phase cells is caused by enhanced APC/CCDH1-mediated CtIP degradation and low CDK levels

doi:10.21203/rs.3.rs-2094165/v1

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The suppression of alt-EJ in quiescent G0-phase cells is caused by enhanced APC/CCDH1-mediated CtIP degradation and low CDK levels

https://doi.org/10.21203/rs.3.rs-2094165/v1

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Alt-EJ is an error-prone DNA double-strand break (DSBs) repair pathway coming to the fore when first-line repair pathways, c-NHEJ and HR, are defective or fail. It is thought to depend on DNA end-resection: a process whereby 3’ single-stranded DNA-tails are generated, initiated by the CtIP/MRE11-RAD50-NBS1 (MRN) complex and extended by EXO1 or the BLM/DNA2 complex. The connection between alt-EJ and resection remains incompletely characterized. Alt-EJ depends on cell cycle phase, is at maximum in G₂-phase, substantially reduced in G₁-phase and almost undetectable in quiescent, G0-phase cells. The mechanism underpinning this regulation remains uncharacterized. Here, we compare alt-EJ in G₁- and G₀-phase cells exposed to ionizing radiation (IR) and identify CtIP-dependent resection as the key regulator. Low levels of CtIP in G₁-phase allow modest resection and alt-EJ as compared to G₂-phase cells. Strikingly, CtIP is undetectable in G₀-phase cells owing to APC/C mediated degradation. Suppression of CtIP degradation with bortezomib or CDH1 knockdown rescues CtIP and alt-EJ in G₀-phase cells. CtIP activation in G₀-phase cells also requires CDK-dependent phosphorylation by any available CDK, but is restricted to Cdk4/6 at the early stages of the normal cell cycle. We propose that suppression of alt-EJ in G₀-phase enhances genomic stability in the large non-cycling cell fraction of higher eukaryotes.

Ionizing radiation

DNA repair

Repair of DNA double strand breaks

Alt-EJ

DNA end-resection

CtIP

APC/C

CDKs

RPA

pulsed-field gel electrophoresis

Exposure of cells to ionizing radiation (IR) induces DNA double-strand breaks (DSBs) that challenge genomic stability. In higher eukaryotes, DSBs are mainly processed by classical non-homologous end-joining (c-NHEJ) and homologous recombination (HR), while alternative end-joining (alt-EJ) and single strand annealing (SSA) contribute variably depending on several parameters including cell cycle phase and growth state ^1,2. While HR is conceptually designed to faithfully restore the genome ², c-NHEJ effectively rejoins DNA ends without template requirements and is therefore less faithful than HR. However, its simplicity and speed make it the most prominent route of DSB repair in higher eukaryotes, especially at high doses of IR ^3,4,5,6. Failures of c-NHEJ or HR may be covered by alt-EJ ^6,7,8, which is slow and more error-prone than c-NHEJ, and a frequent source of chromosomal translocations. This defines it as a mutagenic route of DSB processing that promotes genomic instability ⁹.

How c-NHEJ and HR suppress error-prone alt-EJ to maintain genomic stability is a subject of intensive research. However, emphasis is presently placed on the regulation of DNA end resection (henceforth here simply, resection). Resection, processes the end of a DSB to expose a 3’ single-stranded DNA that can invade a homologous DNA molecule or anneal with another resected end using homologies, and is key to DSB repair pathway choice. Resection inhibits c-NHEJ and triggers homology-directed DSB repair pathways, including HR and SSA. Lower degrees of homology are utilized by alt-EJ ¹⁰. Resection requires CtIP ¹¹ to stimulate the endonuclease activity of MRE11 and proceeds bi-directionally ^12,13. The activity of MRE11-RAD50-NBS1 (MRN) complex process the DNA end in the 3ʹ–5ʹ direction, while Exonuclease 1 (EXO1) or Bloom helicase (BLM)/DNA Replication Helicase/Nuclease 2 (DNA2) catalyze long-range 5ʹ–3ʹ resection ¹⁴. Resection is regulated in a cell cycle-dependent manner and is maximally active in G₂-phase cells, with significantly reduced levels in G₁–phase ^15,16.

Appropriately designed reporter assays monitoring GFP-expression after processing of I-Sce I induced DSBs by a specific DSB repair pathway are frequently used to characterize alt-EJ ^10,17,18. Such studies have reported that Mre11 is required for the resection of as few as 20 bp during alt-EJ ¹⁰ and similar conclusions have been drawn for CtIP and NBS1 ^{10,19,20,21,22}. However, in these reporter assays microhomology is built-in, which may overestimate the contributions of resection on alt-EJ. Pulsed-field Gel Electrophoresis (PFGE) is a technique widely used to study alt-EJ in cells with defects in c-NHEJ ^23,24. Using PFGE, we have recently shown that CtIP depletion suppresses alt-EJ in both exponentially growing and G₂-phase-enriched cells ²⁵.

One key characteristic of alt-EJ is its crucial dependence on the state of growth of the tested cells. Thus, alt-EJ activity is low during G₁-phase and steadily increases as cells move to G₂-phase. Notably, alt-EJ is nearly completely abrogated in some cell lines in G₀-phase ^24,26,27. Epidermal growth factor receptor (EGFR) inhibition in proliferating cells suppresses alt-EJ only marginally, and addition of EGF in G₀-phase cells increases alt-EJ marginally, which suggests that inhibition of alt-EJ in G₀ cells is not a direct consequence of suppressed growth factor signaling ²⁸. While the mechanism underlying the suppression of alt-EJ in G₀-phase cells remains to be elucidated, a tenable hypothesis is that suppression of alt-EJ derives from a reduction in resection in G₀, which immediately raises the question as to the underpinning mechanism.

The cell-cycle-dependent regulation of resection is achieved primarily through the CDKs ²⁹. CtIP phosphorylation by CDKs on Thr847 and Ser327 critically regulates resection in vertebrates ^30,31,32. Interestingly, CDK2-dependent phosphorylation of Ser276 and Thr315 also promotes CtIP binding to PIN1 to dampen resection ³³. CDKs also promote resection by phosphorylating EXO1 ³⁴, NBS1 ^35,36,37 and DNA2 ³⁸. Finally, CDKs regulate resection by suppressing resection-blocks raised by 53BP1 and DNA helicase B (HELB) ^39,40,41.

The oscillating activity of CDKs throughout the cell cycle is regulated by the periodic degradation by the ubiquitin–proteasome system of cyclins and CDK inhibitors (CKIs) ⁴². Central in this process is a pair of RING-type E3 ubiquitin ligases: SCF (for SKP1/Cullin/F-box protein) and the anaphase-promoting complex/cyclosome (APC/C) ⁴³. SCF remains active from late G₁- to late G₂-phase and selectively degrades proteins through priming for degradation. S-phase kinase-associated protein 2 (SKP2) is the main substrate recognition factor of SCF ⁴³. CDC20 homologue-1 (CDH1) is the main substrate recognition factor of the APC/C complex, which is activated in late M- to early/mid-G₁-phase and retains activity until inception of DNA replication ^43,44.

In addition, APC^CDH1 regulates resection by regulating cyclin and CtIP abundance. IR activates APC^CDH1 and mediates CtIP degradation to limit resection in G₂-phase cells ⁴⁵. In addition, APC^CDH1 competes with SCF and indeed SKP2 depletion causes the activation of APC^CDH1 and the degradation of CtIP that compromises alt-EJ in G₂-phase-enriched cells ²⁵. These results suggest that the regulation of alt-EJ is achieved through CDK-dependent activation of key components of the resection apparatus and the SCF-APC/C dependent regulation of their levels by protein degradation. The regulation of alt-EJ by CtIP levels and its CDK dependent activation remains to be elucidated in G1- and particularly in G₀-phase cells.

In the present study we continue the elucidation of the mechanistic regulation of alt-EJ by focusing on cells of G₁- and G₀ -phase. Since HR is suppressed in this phase of the cell cycle, it is possible to rather specifically investigate the role of resection in alt-EJ ^10,46,47,48. We report that CtIP is the main regulator of alt-EJ activity in G₁-phase and that its degradation or reduced phosphorylation abrogates resection and alt-EJ. Interestingly, G₀-phase cells are more radioresistant than G₁-phase phase cells, which suggests that suppression of alt-EJ improves genomic stability. Since almost all terminally differentiated cells in the human body are non-cycling and stem cells frequently rest in G₀-phase, it is possible that suppression of error-prone alt-EJ is a mechanism for improved genome stability and tumor suppression.

Cell culture and irradiation

Human normal fibroblasts, 82-6 hTert, and human lung carcinoma cells, A549, were grown and were irradiated as reported earlier ^15,25.

Survival determination

Cell survival was measured using the colony formation assay as described previously ²⁵.

RNA interference

Knockdown experiments were carried out using specific siRNAs against the proteins of interest. The siRNAs (Table S1) were delivered to the cells by nucleofection using the Nucleofector-2B device (Lonza), as described previously ^15,25.

Treatment of cell with inhibitors

All inhibitors (Table S2) were added 1h before irradiation unless indicated otherwise.

Flow cytometry (FC) analysis of resection

Resection was analyzed by FC using RPA70 detection, as described earlier ^15,25. Antibodies used are listed in Table S3. Experiments were replicated 3 times and representative histograms from one experiment are shown.

Polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting

SDS-PAGE and immunoblotting were employed as described ^15,25. Antibodies used are listed in Table S3.

Real-time polymerase chain reaction (PCR)

mRNA levels of CtIP were determined by real-time PCR. We used commercially available kits to extract total RNAs (Roche, 11828665001) and synthesize the first cDNAs strand (Thermo fisher scientific, K1631). The PCR conditions and primers used are listed in Table S4 and 5, respectively. Data is analyzed using Light-Cycler software Version 4.1 (Roche).

Pulsed-field gel electrophoresis (PFGE)

To analyze alt-EJ, PFGE was performed as described earlier ²⁵.

G1-phase cell synchronization

Thymidine combined with a nocodazole block were used to synchronize exponentially growing cells in G₁-phase. Cells were incubated overnight (~16h) in medium containing 2mM thymidine. Subsequently, cells were incubated in fresh growth medium for 4h before adding nocodazole at 40ng. After another 9h, cells were released from mitosis by gently shaking flasks or dishes and were used 4h later as they entered early G₁-phase.

Statistical Analyses

Results are expressed as mean±standard error (SE) calculated from three of more repeats. Statistical significance between experimental groups was determined by t-test. The significance of differences between individual measurements is indicated by a symbol: * p<0.05, ** p<0.01, N.S. non-significant.

Alt-EJ is suppressed in G₀- as compared to G₁-phase cells

We previously demonstrated that alt-EJ is suppressed in mouse and hamster c-NHEJ mutants when they grow into a plateau- and enter G₀-phase ^26,49,50. Here we extend these observations to human cells and begin the elucidation of the underpinning mechanisms. Figure 1a shows that under the conditions employed, 82 − 6 hTert cells grow logarithmically for 3 days and enter later, if not refed, a plateau-phase. In plateau-phase, the majority of cells accumulate with a G₁–phase-equivalent DNA content (Fig. 1b). The parallel decrease in Ki67 level (Fig. 1b), a cell proliferation marker ^51,52,53 that is low in G₀ cells, shows that plateau-phase cultures mainly comprise G₀-phase cells. To reduce irreproducibility associated with unfed plateau-phase cultures ¹⁵, we generated G₀-phase 82 − 6 hTert cell populations using serum deprivation (SD) starting at day-2 of growth (Fig. 1a). Figures 1a and 1b show good stability and enrichment in G₀-phase similar to that of plateau-phase cultures at day-3. SD-cultures are therefore exclusively used in the experiments. To compare the responses of G₀- to those of G₁–phase cells, we employed the protocol outlined in Figure S1a to obtain enriched populations of G₁–phase cells. This protocol generates highly enriched G₁–phase cells, 4 h after release from nocodazole-block (Figure S1b).

Analysis of DSB repair using PFGE after exposure to 20Gy (Fig. 1c) shows indistinguishable repair potential in exponentially growing, as well as in enriched G₀- and G₁-phase 82 − 6 hTert cells (see Fig. 1d for a confirmation of cell cycle distribution and growth state). This repair activity mainly reflects the function of c-NHEJ ^5,54, which removes over 90% of DSBs within 2h independently of cell growth and cell cycle stage. Treatment of cells with the DNA-PKcs inhibitor NU7441 (2.5 µM, DNA-PKcsi) inhibits c-NHEJ and allows alt-EJ to come to the fore ⁶. Exponentially growing cells treated with NU7441 show a profound reduction in DSB repair, but alt-EJ still repairs over 50% of DSBs within 2 h; G₁-phase cells show similar repair activity in the presence of DNA-PKcsi. Strikingly, G₀-phase cells treated with DNA-PKcsi are profoundly deficient in DSB repair, particularly at early times after IR and repair less than 20% of induced DSBs in 4h (Fig. 1c). This is an evidence for a strong suppression of alt-EJ, as cells transit from G₁ to G₀-phase of the cell cycle.

Lung adenocarcinoma A549 cells can also be maintained under analogous growth conditions to generate highly enriched G₀- and G₁-phase cultures (Figure S1c and d). When DSB repair is analyzed in the presence of DNA-PKcsi, a similar, strong suppression of alt-EJ is observed in G₀-phase cells (Figure S1e and f). We conclude that suppression of alt-EJ in G₀-phase cells is a general phenotype, detectable in many cell lines from different species, and focus below on the elucidation of the underpinning mechanisms.

The results presented here and the majority of those reported earlier ^26,49,50, analyze DSB repair at high doses of IR. We recently reported profound adaptations in DSB repair pathway choice with increasing IR up to 20 Gy ^5,55,56,57. We therefore examined the above described suppression of alt-EJ in human G₀-cells after exposure to 2Gy using γH2AX foci scoring (results not shown). While strongly suppressed DSB repair activity was observed in G₀-phase cells, the difference from G₁-phase cells was small, because G₁-phase cells show limited repair activity after treatment with NU7441. We investigate whether this reduced growth-state effect on alt-EJ at low IR doses reflects the earlier discussed ⁵⁸ divergence between DSB repair analysis with assays of physical DNA integrity versus resulting DDR, or the dose-dependence of alt-EJ ^59,60. Therefore, we focus here on effects measured after exposure to high IR doses.

DNA end-resection is undetectable in G₀-phase cells

As resection facilitates alt-EJ, we investigated whether a reduction underpins its suppression in G₀– versus G₁-phase cells. We employed a previously described method ^5,55,56,57 enabling cell-cycle-specific analysis in G₀- and G₁-phase, as outlined in Figure S2a and b. Figure 2a shows as already reported ¹⁶ low-level resection in G₁-phase cells of exponentially growing cultures (the method of analysis obviates synchronization in G₁-phase and is free of manipulations) after exposure to 10Gy. In accordance with recent results in G₂-phase ^56,57, NU7441 fails to modify resection in G₁-phase (Figure S2c). Therefore, subsequent resection experiments are carried out in the absence of inhibitor. The same pattern of resection is also observed in A549 cells with low but detectable resection in G₁-phase cells of exponentially growing cultures after exposure to 10Gy (Figure S2d) that remains unaffected by NU7441 (Figure S2e).

Notably, similar analysis in G₀-phase in both cell lines, fails to detect resection (Fig. 2a and Figure S2d). These observations suggest that the reduced alt-EJ in G₀- versus G₁-phase cells derives from a reduction in resection activity. Because resection in G₁-phase cells is relatively small (Fig. 2a), we sought additional experimental validation of the putative connection between resection and alt-EJ activity.

CtIP levels are undetectable in G₀-phase cells

The undetectable resection in G₀-phase cells may derive either from a reduction in the abundance of components of the resection machinery, or from a down-regulation of their activities. We measured therefore the levels of key proteins of the resection apparatus in exponentially growing, as well as in G₀- and G₁-phase 82 − 6 hTert cells, before and 1 or 3 h after exposure to 10 Gy. Figure 2b shows that while MRE11, RAD50, NBS1 and DNA2 show only minor fluctuations, CtIP and EXO1 are markedly reduced in G₀-phase cells, as compared to G₁-phase or to exponentially growing cells. Irradiation profoundly increases the levels of CtIP in exponentially growing and G₁-phase cells, but CtIP, or a radiation-induced increase, are undetectable in G₀-phase cells. Because CtIP is the central regulatory component of the resection apparatus, its depletion in G₀-phase cells suggests a role in resection that is also undetectable.

The reduction of CtIP-protein levels in G₀-phase cells may reflect the normally occurring reduction in gene expression during quiescence. We measured therefore the mRNA levels of CtIP in exponentially growing, as well as in G₁- and G₀-phase cells. Figure 2c shows no significant differences in mRNA levels in the different conditions analyzed. Therefore, we turned our attention to CtIP stability. Figure 2d shows successful depletion of CtIP, EXO1 and DNA2 in exponentially growing 82 − 6 hTert cells and Fig. 2e shows that CtIP and DNA2 depletion suppresses resection in G₁-phase, whereas EXO1 depletion is ineffective. Also, inhibition of MRE11 endonuclease activity with PFM01 inhibits resection, whereas Mirin, an inhibitor of MRE11 exonuclease activity ¹² is ineffective (Fig. 2e). Figure S3a summarizes results from several experiments and confirms these conclusions.

To directly connect resection with alt-EJ, we analyzed G₁-phase cells after depletion of CtIP, EXO1 or DNA2, or after inhibition of MRE11 activity. The results in Fig. 3a show that CtIP knockdown not only suppresses resection profoundly, but also alt-EJ; while EXO1 knockdown that has no effect on resection, leaves alt-EJ unaffected. In this experiment, DNA2 knockdown has no effect on alt-EJ, which might suggest that short range resection in G₁ phase cells is enough for alt-EJ. PFM01 that strongly suppresses resection, suppresses alt-EJ as well, whereas Mirin remains ineffective on both endpoints. We conclude that in G₁-phase cells alt-EJ benefits from resection initiated by the MRN/CtIP and that the depletion of CtIP in G₀-phase cells causes the suppression of alt-EJ observed. But how is CtIP depleted in G₀-phase cells?

APC^CDH1 depletes CtIP in G₀-phase cells

APC^CDH1 ubiquitinates CtIP and mediates its degradation by the proteasome ^33,45,61. We examined therefore the effect of the proteasome inhibitor bortezomib on CtIP levels. Treatment of G₀-phase 82 − 6 hTert cells with 2 µM bortezomib for 2h markedly increases CtIP levels (Fig. 3b). Moreover, CDH1 knockdown similarly upregulates CtIP levels (Fig. 3b). Finally, in A549 CDH1^-/- cells ²⁵, CtIP is clearly detectable in G₀-phase and increases further in G₁-phase cells, as expected, after suppression of CtIP degradation (Fig. 3c). These results show that increased APC/C^CDH1 activity in G₀-phase cells causes the degradation of CtIP.

We investigated whether suppression of CtIP degradation using the above treatments and conditions restores alt-EJ and resection in G₀-phase cells. Figures 3e shows that CDH1 depletion (Fig. 3d), or treatment with bortezomib restore to a considerable degree alt-EJ in G₀-phase cells, without affecting their cell cycle distribution (Figure S3b). However, neither CDH1 depletion nor Bortezomib detectably elevate resection in G₀ phase cells (Figure S3c and d). We discuss this result further below. Moreover, alt-EJ remains at G₁-phase levels when A549 CDH1^-/- cells enter G₀-phase (Fig. 3f). Collectively, the above results identify APC/C as the key regulator of CtIP levels throughout the cell cycle that directly regulate resection and alt-EJ activity.

Low CDK activity in G₀-phase cells keeps CtIP inactive and alt-EJ suppressed

CtIP activity is also regulated by CDK-dependent phosphorylations ^{29,30,31,32,33}. High CDK activity is a feature of proliferating cells and is low in G₀-phase cells ^62,63. When mitotic cells divide and enter G₁-phase, or when G₀-phase cells are stimulated to proliferate by growth factors, CDK4/6 is activated by D-type cyclins, whose expression is growth factor inducible ⁶⁴. In late G₁-phase, Cyclin D-CDK4/6 complexes phosphorylate pocket proteins to release E2F transcription factors to induce the transcription of G₁/S target genes, including the one encoding for cyclin E, thus activating CDK2/Cyclin E ⁶⁵.

We considered the possibility that active CDK4/6 in G₁-phase cells phosphorylates CtIP, and that this phosphorylation is important for CtIP activation, and thus for resection and alt-EJ. We tested, therefore, the effects of CDK inhibition on resection and alt-EJ in enriched G₁-phase 82 − 6 hTert cells. Figure 4a shows that CDK4/6 inhibition markedly suppresses alt-EJ in G₁-phase (see Fig. 4b for cell cycle distribution) to levels similar to those of G₀-phase cells, whereas inhibition of CDK2 or CDK1 has no effect. This can be rationalized by CDK4/6 being fully active, but CDK1 and CDK2 still remaining inactive at this stage in G₁-phase. This leaves CDK4/6 as the sole cell cycle kinase able to activate CtIP. Figure 4c shows that CDK4/6 inhibition also suppresses resection in G₁-phase cells, while inhibition of CDK2 or CDK1 only has a small effect on resection. Figure 4d confirms that CDK4/6 inhibition suppresses radiation-induced CtIP phosphorylation at threonine 847 (CtIP-T847), as well as the radiation induced stabilization of the protein. It has been reported using confluent (practically G₀-phase) 82 − 6 hTert cultures that PLK3 phosphorylates and activates CtIP for resection supporting a special form of resection-dependent c-NHEJ ⁶⁶. We investigated therefore the effects of PLK3 inhibition on the regulation of G₁-phase alt-EJ and resection. PLK3 inhibition using GW843682X (2µM) fails to suppress alt-EJ (Figure S4a) or resection (Figure S4b) in G₁-phase 82 − 6 hTert cells. Thus, PLK3 may be specifically utilized in G₀-phase cells to regulate resection-dependent c-NHEJ, when CDK4/6 activity is low and alt-EJ suppressed.

We investigated how inhibition of CDKs affects alt-EJ in WT and CDH1^−/− A549 cells, tested in G₁- and G₀-phase. Notably, similar to 82 − 6 hTert cells, alt-EJ only depends on CDK4/6 in WT enriched G₁-phase A549 cells. In contrast, in CDH1^−/− A549 cells, single inhibition of CDK4/6, CDK1 or CDK2 exerts only small inhibitory effects on alt-EJ. However, a cocktail including all inhibitors causes a strong suppression of alt-EJ, both in enriched G₁– as well as G₀-phase cells (Fig. 5a). This shift in the absolute dependence of alt-EJ on a specific set of CDKs likely reflects the general stabilization of Cyclins in G₁-phase following APC/C^CDH1 inactivation, which leaves several CDKs active to interchangeably activate CtIP. Indeed, Fig. 5b shows that in CDH1^−/− A549 cells, cyclins A2 and B1 are stabilized, while the levels of cyclin D1 and cyclin E1 remain unaffected (Figure S3e). The same trends are also observed in 82 − 6 hTert cells after CDH1 knockdown (Figs. 5c and S3e). Thus, CDH1 depletion shifts CDK activity in early G₁-phase from a specific CDK4/6 activation to a general CDK4/6, CDK1 and CDK2 activation that explains the results obtained. The degradation specifics by APC/C of cyclins and CtIP in G₁-phase, allow CDK-dependent CtIP activation in G₁ -phase of the cell cycle and thus the resection and alt-EJ activities observed.

Suppression of alt-EJ in G₀-phase cells enhances genomic stability

The above results uncover a well-designed, programmed suppression of alt-EJ in G₀-phase cells that is part of a more general cell cycle regulation of CtIP levels and activity, which in-turn regulate resection and alt-EJ. But, are there benefits for the genomic stability of irradiated cells from such regulatory adaptations of the activities of specific DSB repair pathways? It is relevant that alt-EJ is error-prone and therefore a major source of genomic instability. To address this question, we exposed cells to IR, either in G₀- or G₁-phase and plated them at lower density to allow for colony formation 6h later. Figure 5e shows that in both cell lines, G₀-phase cells are significantly more radioresistant than G₁-phase cells, which confirms survival benefits from alt-EJ suppression.

CtIP levels regulate resection and alt-EJ throughout the cell cycle

We show above that CtIP cooperates with MRE11 to regulate resection in G₁-phase cells. Indeed, CtIP may be involved in the regulation of alt-EJ throughout the cell cycle in two distinct ways: First, CtIP levels fluctuate throughout the cell cycle and are, as we show here, undetectable in G₀-phase phase owing to the activity of APC^{CDH1 45} that mediates nearly complete CtIP degradation. The inactivation of APC/C^CDH1, as cells progress from G₀- to G₁- and then to S-phase ^45,67, allows CtIP levels to increase, reaching a maximum at the end of G₂-phase. Notably, our past and present work shows that alt-EJ is almost fully suppressed in G₀-phase and fully active in G₂-phase ^24,26,27. It remains to be investigated whether residual alt-EJ activity in G₀-phase cells reflects residual CtIP activity, or resection-independent alt-EJ. Second, CtIP activity is regulated by CDKs ^30,31,32,33: CDK activity is at its lowest level in quiescent cells, increases in G₁-phase cells, and is again highest in G₂-phase cells ⁶⁸. APC/C^CDH1 activity marks for degradation, in addition to CtIP, also cyclins and suppresses thus the CDK activity that is required for CtIP activation ⁶⁹. This parallel evolution of ubiquitination and phosphorylation activities ensures that when CtIP is present, there will be CDK activity to activate it. It also explains why CDH1 deficiency or treatment with bortezomib have effects on both of these endpoints.

CtIP-stability regulation during the cell cycle and in response to DNA damage

HR is conceptually designed to faithfully restore the genome and to maintain thus genomic stability. CtIP-mediated resection exposes a 3’ single-stranded DNA that invades the sister chromatid during HR. In G2-phase cells, APC/C^CDH1 mediates the clearance of CtIP from DSBs through ubiquitin-mediated degradation, thus limiting resection and adjusting it to HR requirements ⁴⁵. We show here that CtIP is involved in alt-EJ in G₁-phase cells, which actually promotes genomic instability (Fig. 5d). CtIP is stabilized by IR in G₁-phase cells (Fig. 2b), which is actually opposite to the destabilization induced in G₂-phase. This suggests benefits from CtIP stabilization, possibly at the expense fidelity, at least when c-NHEJ is inactive. Indeed, CtIP mutated chicken B cells (DT40) cells are sensitive to IR in G₁-phase ⁷⁰. On the other hand, CtIP depletion rescues the DSB repair defect of Artemis mutants in G₁-phase human fibroblasts, while it produces no significant effects in wild-type cells. This suggests that CtIP depletion causes a switch from resection-dependent to resection-independent DSB processing in G₁-phase cells ¹⁶. Taken together, these data suggest that CtIP is involved in the repair of DSBs throughout the cell cycle, but its regulation and roles have a strong cell cycle component.

Relevance of alt-EJ suppression to radiotherapy and carcinogenesis

Processing of DSBs by alt-EJ generates chromosomal translocations that cause genomic instability and cancer ⁹. DSBs naturally occur in a cell in the order of 10 to 50 DSBs per cell per day, depending on cell cycle and tissue ⁷¹. Most recent estimates put the number of cells in an average human body at around 3x10¹⁴, most of which are non-cycling. These numbers indicate that a very high number of DSBs are induced every day in the human body. Therefore, protection from carcinogenesis may be achieved by suppressing mutagenetic repair of spontaneous DSBs in the majority of cells.

On the other hand, tumor recurrence after radiation treatment is thought to be caused by radioresistant cells, which may derive from the pool of G₀-phase cancer cells, which as we show are radioresistant. Here, the suppression of alt-EJ in quiescent cells might contribute to tumor recurrence by increasing the survival of tumor cells. Collectively, we conclude therefore that the default regulation of alt-EJ by CtIP suppresses carcinogenesis, but may contribute to tumor recurrence.

alt-EJ benefits from resection in cells of G₁-phase in case of c-NHEJ suppression

The initiation of 5'-3' resection at DNA ends is a critical determinant of repair pathway choice, as it commits DSBs to HR while suppressing c-NHEJ ^46,48,72. Until recently, it was thought that resection only occurs in S- and G₂- phase cells, and that it was inhibited in G₁-phase by the Ku70/80 heterodimer and other proteins ⁴⁶. However, RPA loading at DSBs is indeed observed in irradiated G₁-phase cells, suggesting limited resection ^48,73,74. It has been suggested that in G₁-phase, resection is dependent on DSB number, with three or less endonuclease-induced DSBs per cell being insufficient and four or more sufficient to activate resection ⁷⁵. Notably, resection has been reported in G₁-phase human cells, particularly for complex DSBs induced by high LET particles, or at high doses of X-rays ^66,76. Interestingly, 2 Gy of α-particles, induced G₁-resection-dependent DSB repair that is independent of PARP1 and LIG1/3, but dependent on Artemis and DNA-PKcs. Because DNA-PKcs inhibition leaves unchanged the repair kinetics of Artemis deficient cells, it was concluded that this form of resection-dependent DSB repair reflects special forms of c-NHEJ, rather than alt-EJ ¹⁶.

We have previously demonstrated the suppression of alt-EJ when cells enter G₀-phase ^26,49. Here we extend these studies to human cells, uncover the role of resection in the regulation of alt-EJ, and correlate the suppression of alt-EJ with compromised resection in G₀-cells. Taken together, the above discussed reports and our present results show that resection can be induced in G₁-phase, even in G₀-phase cells by complex DSBs, to facilitate special forms of c-NHEJ ¹⁶. On the other hand, simple DSBs in a c-NHEJ deficient background, activate resection specifically in G₁-phase that supports alt-EJ.

Collaboration between APC^CDH1 and CDKs in the regulation of DSB repair

CDK1 and CDK2 phosphorylate CtIP, NBS1, EXO1 and DNA2 to regulate resection ^77,78,79 in G2-phase cells. Moreover, the necessity of CDK2-dependent CtIP phosphorylation on Thr847 is known for resection in G₁-phase cells. However, here using appropriately synchronized cells, we provide evidence for a contribution of CDK4/6 to CtIP activation and resection in G₁-phase of cells. Thus, it seems plausible that all cell cycle CDKs also activate CtIP.

In addition to phosphorylation-dependent regulation of CtIP activity, protein levels are also directly regulated and modulate resection at DSBs. APC/C^CDH1 targets CtIP for proteasomal degradation after mitotic exit to generate the low resection environment that persists during G₁-phase ⁴⁵. Moreover, it is well accepted that oscillating activity of CDKs throughout the cell cycle is regulated by the periodic degradation of cyclins and CDK inhibitors (CKIs) by the ubiquitin–proteasome system ⁴², in which APC/C^CDH1 complex plays a central role, as it targets cyclin B1 and cyclin A2 for degradation. On the other hand, the affinity of CDH1 towards APC/C is suppressed by CDK phosphorylation ^43,44. These feedback mechanisms define the cross talk between APC^CDH1 and CDKs in the regulation of the cell cycle with extensions to DSB repair. These findings place the ubiquitination pathway and particularly APC^CDH1 in an intermediate node in the DSB repair pathway choice network.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

AVAILABILITY OF DATA AND MATERIALS

As requested.

ACKNOWLEDGEMENTS

The authors are indebted to Drs. M. Löbrich and A. Sartori for providing cell lines and reagents. Work supported by grants from BMBF (02NUK037B, 02NUK043B and 02NUK054B), DFG (IL51.10, IL51.11 and GRK1739), BMWi (50WB1836) and DAAD Project #57515880.

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The suppression of alt-EJ in quiescent G0-phase cells is caused by enhanced APC/CCDH1-mediated CtIP degradation and low CDK levels

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Alt-EJ is suppressed in G₀- as compared to G₁-phase cells

DNA end-resection is undetectable in G₀-phase cells

CtIP levels are undetectable in G₀-phase cells

APC^CDH1 depletes CtIP in G₀-phase cells

Low CDK activity in G₀-phase cells keeps CtIP inactive and alt-EJ suppressed

Suppression of alt-EJ in G₀-phase cells enhances genomic stability

Disscussion

CtIP levels regulate resection and alt-EJ throughout the cell cycle

CtIP-stability regulation during the cell cycle and in response to DNA damage

Relevance of alt-EJ suppression to radiotherapy and carcinogenesis

alt-EJ benefits from resection in cells of G₁-phase in case of c-NHEJ suppression

Collaboration between APC^CDH1 and CDKs in the regulation of DSB repair

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

The suppression of alt-EJ in quiescent G0-phase cells is caused by enhanced APC/CCDH1-mediated CtIP degradation and low CDK levels

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Alt-EJ is suppressed in G0- as compared to G1-phase cells

DNA end-resection is undetectable in G0-phase cells

CtIP levels are undetectable in G0-phase cells

APCCDH1 depletes CtIP in G0-phase cells

Low CDK activity in G0-phase cells keeps CtIP inactive and alt-EJ suppressed

Suppression of alt-EJ in G0-phase cells enhances genomic stability

Disscussion

CtIP levels regulate resection and alt-EJ throughout the cell cycle

CtIP-stability regulation during the cell cycle and in response to DNA damage

Relevance of alt-EJ suppression to radiotherapy and carcinogenesis

alt-EJ benefits from resection in cells of G1-phase in case of c-NHEJ suppression

Collaboration between APCCDH1 and CDKs in the regulation of DSB repair

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Alt-EJ is suppressed in G₀- as compared to G₁-phase cells

DNA end-resection is undetectable in G₀-phase cells

CtIP levels are undetectable in G₀-phase cells

APC^CDH1 depletes CtIP in G₀-phase cells

Low CDK activity in G₀-phase cells keeps CtIP inactive and alt-EJ suppressed

Suppression of alt-EJ in G₀-phase cells enhances genomic stability

alt-EJ benefits from resection in cells of G₁-phase in case of c-NHEJ suppression

Collaboration between APC^CDH1 and CDKs in the regulation of DSB repair