TORC2 inhibition triggers yeast chromosome fragmentation through misregulated Base Excision Repair of clustered oxidation events

Abstract

Combinational therapies provoking cell death are of major interest in oncology. Combining TORC2 kinase inhibition with the radiomimetic drug Zeocin results in a rapid accumulation of double-strand breaks (DSB) in the budding yeast genome. This lethal Yeast Chromosome Shattering (YCS) requires conserved enzymes of base excision repair. YCS can be attenuated by eliminating three N-glycosylases or endonucleases Apn1/Apn2 and Rad1, which act to convert oxidized bases into abasic sites and single-strand nicks. Adjacent lesions must be repaired in a step-wise fashion to avoid generating DSBs. Artificially increasing nuclear actin by destabilizing cytoplasmic actin filaments or by expressing a nuclear export-deficient actin interferes with this step-wise repair and generates DSBs, while mutants that impair DNA polymerase processivity reduce them. Repair factors that bind actin include Apn1, RFA and the actin-dependent chromatin remodeler INO80C. During YCS, increased INO80C activity could enhance both DNA polymerase processivity and repair factor access to convert clustered lesions into DSBs.

Fig. 1. TORC2 inhibition and Ogg1 activity convert Zeocin-generated DNA base oxidation into DSB without DSB repair machinery or replication.

a, b Massive yeast chromosome fragmentation occurs in G1-arrested as well as exponential cultures. Wild-type (WT, GA-1981) and an isogenic rad51 lig4 double mutant (GA-6098) were grown exponentially (a), or arrested in G1 by α-factor for 1.5 h (b). Cells were treated with 50 μg/ml Zeocin, 1 mM H₂O₂, or 0.05% MMS alone, or in combination with the potent TORC2 inhibitor CMB4563 (CMB; Supplementary Fig. 1a) at 0.5 μM for 80 min. Genomic DNA was isolated in an agarose plug at 30 °C as described in Methods, chromosomes were separated on a non-denaturing CHEF gel and stained with SYBR safe. The signal intensity of each lane was measured, and ratios of the background-depleted signal of B (from 0.56 Mbp down to ~20 kbp) over A (above 0.57 Mbp, or from the Chr VIII/V band through largest Chr IV/XII) are indicated below the image (data summarized in Supplementary Data 1; and FACS analyses in Supplementary Fig. 5). c To obtain a given mean fragment size (x-axis), the mean number of randomly occurring breaks for each chromosome was calculated and plotted along the y-axis from Chr I to Chr XVI, as described in Supplementary Fig. 2, legend. The black line represents the number of DSBs per genome needed to generate the indicated fragment size. d Theoretical mean number of randomly distributed DSB per chromosome for a mean fragment size of 100 kb was determined by 1000 repeat calculations as in panel c. The black bars indicate a 20% error in the estimated mean fragment size (measured mean fragment size ranges from 80 to 120 kb under standard conditions). e Zeocin treatment induces substrate for hOGG1-mediated linearization of supercoiled plasmids in vitro. Supercoiled plasmid DNA was mixed with the indicated concentration of Zeocin for 60 min at 37 °C, in the indicated molar ratio, and then incubated with or without purified hOGG1 for 30 min at 37 °C. Plasmid DNA was recovered and analyzed on an agarose gel to separate nicked, linearized, and supercoiled forms. The signal intensity of plasmid forms was quantified by ImageJ, and their ratio was plotted below the gel.

Fig. 2. BER N-glycosylase enzymes and AP nuclease activities are necessary for YCS.

a Scheme of short-patch (SP) and long-patch (LP) BER pathways with yeast enzymes in lower case and mammalian enzymes in capitals based on recent reviews^,. In SP-BER, the equivalence of yeast Pol4 and mammalian POLβ is unclear (see text). b Loss of N-glycosylase activity confers resistance to YCS. Exponentially growing wild-type (GA-8369) and Ogg1-deg ntg1 ntg2 (GA-8457, NGΔ) cells were treated with 0.5 mM indole-acetic acid (IAA) for 4 h to provoke degradation of Ogg1 (Supplementary Fig. 3a shows proof of degradation). Cells were then treated with DMSO alone, and either NVS-BHS867, Zeocin, or both drugs, as indicated, for 70 min. Genomic DNA was analyzed on a CHEF gel, visualized using SYBR safe, and chromosome integrity was quantified as in Fig. 1a and summarized in Supplementary Data 1. c Loss of AP endonuclease activity renders cells resistant to YCS. Exponentially growing wild-type (GA-8369) and apn1 APN2-deg RAD1-deg (GA-8509, APΔ) cells were treated with 0.5 mM IAA for 1 h to deplete Apn2 and Rad1. Cells were then incubated with 0.5 μM CMB4563 in the presence of Zeocin as indicated for 70 min and analyzed as in (b).

Fig. 3. Impaired processivity of DNA Pol δ and Pol ε but not Pol4 or Rev3, reduces YCS efficiency.

a Strains lacking DNA Pol δ subunit Pol32 are partially resistant to YCS. Wild-type (GA-1981), pol4Δ (GA-10595), pol32Δ (GA-9686), and pol4Δ pol32Δ (GA-10697) mutants were exponentially cultured in SC medium. Cells were treated with 0.5 μM CMB4563, 50 μg/ml Zeocin, or a combination of both reagents for 60 min. Chromosomal integrity was analyzed and quantified as in Fig. 1a. b As 3a, except wild-type (WT, GA-1981), and two independent isolates of pol3-ct (pol3-ct-1 and -2; GA-10997 and GA-11000) were exponentially grown in SC medium, and were treated with 0, 50, or 100 μg/ml Zeocin with or without 0.5 μM CMB4563 as indicated for 80 min. Chromosomal integrity was analyzed and quantified as in Fig. 1a and in Supplementary Data 1. c As 3a, except that isogenic wild-type (WT, GA-1981) and rev3Δ (GA-9683) cells were grown exponentially and incubated with the indicated reagents for 80 min, prior to CHEF gel analysis and quantitation as in Fig. 1a and in Supplementary Data 1. d Reduced DNA Pol ε activity confers partial resistance to YCS. Wild-type (GA-741) and pol2-18 (GA-742) cells (see) were exponentially cultured in SC at 25 °C. Cell culture was shifted to 37 °C for 40 min, treated with 1 μM CMB4563, 75 μg/ml Zeocin, or a combination of both reagents for 70 min, prior to CHEF gel analysis and quantitation as in Fig. 1a and in Supplementary Data 1. Partial resistance to chromosome shattering is seen under YCS conditions. e Loss of Trf4 enhances Zeocin sensitivity but reduces YCS. Wild-type (GA-1981) and trf4Δ (GA-10632) cells were exponentially cultured in SC, then treated with 50 μg/ml Zeocin with or without 0.5 μM CMB4563, as above for 80 min, prior to CHEF gel analysis and quantitation. In the absence of CMB, trf4Δ shows increased chromosome fragmentation on Zeocin (B/A = 1.1 vs. 0.3 in WT), but under YCS conditions, trf4Δ is partially resistant (B/A = 4.0 vs. 10 for WT). CHEF gel quantitation as in Fig. 1a and Supplementary Data 1.

Fig. 4. AP endonucleases, but not N-glycosylases, are required for YCS in G1 cells.

a N-glycosylase activity is largely dispensable for YCS in G1-arrested cells. Exponentially growing wild-type (GA-8369) and Ogg1-deg ntg1 ntg2 (NGΔ; GA-8457) cells were treated with 0.5 mM IAA for 2 h (Ogg1 degradation in Supplementary Fig. 3a), then α-factor was added for 2 h to the arrest the culture in G1 (FACS analysis in Supplementary Fig. 5). Cells were then treated with DMSO or CMB4563 in the presence of the indicated amounts of Zeocin for 80 min, prior to CHEF gel analysis and quantitation as in Fig. 1a. b YCS occurs in an AP-endonuclease-dependent manner in G1. Exponentially growing wild-type (GA-8369) and apn1 APN2-deg RAD1-deg (APΔ; GA-8509) cells were treated with 0.5 mM IAA and α-factor for 2 h (FACS analysis in Supplementary Fig. 5; Rad1 degradation control in Supplementary Fig. 3a). Cells were treated with CMB4563 and Zeocin as indicated for 80 min, prior to CHEF gel analysis and quantitation as in Fig. 1a and Supplementary Data 1. c Differential sensitivity of G1 cells and exponential cultures to Zeocin. Wild-type (WT), NGΔ, and APΔ cells were grown in complete minimal media (SC) and arrested in G1 by α-factor together with 0.5 mM IAA for 2 h (FACS analysis in Supplementary Fig. 5). Cells were then treated with 50 μg/ml Zeocin, 0.5 μM CMB4563, or both drugs for 90 min. Cells were spotted on YPAD in a 5-fold dilution series. Images taken after 2 days were quantified for cell survival percentages normalized to the DMSO control. Data points are from 3 biological replicates. d Wild-type, NGΔ, and APΔ cells were exponentially grown in SC and treated with 0.5 mM IAA for 2 h to deplete degron-tagged proteins, and then treated as panel (c). Data points are from 4 biological replicates. AP endonuclease loss increases survival of YCS conditions in G1 arrested cells, consistent with the resistance to chromosome breakage, but not in exponential cultures. N-glycosylase activities (Ogg1, Ntg1, Ntg2) loss aids survival of Zeocin and TORC2 inhibitors in exponentially growing cells, but not in G1-arrested cultures.

Fig. 5. Loss of Trf4 and DNA Pol ε reduce YCS in G1, while Lig1 loss increases it.

a Ablation of POL32 does not confer YCS resistance in G1-arrested cells. Wild-type (WT, GA-1981), pol4Δ (GA-10595), pol32Δ (GA-9686), and pol4Δ pol32Δ (GA-10697) mutants were arrested in G1 by α-factor for 100 min (Supplementary Fig. 5 for FACS). Cells were then treated with 0.5 μM CMB4563, 50 μg/ml Zeocin, or both for 70 min prior to CHEF gel analysis and quantitation as in Fig. 1a; and Supplementary Data 1. b Wild-type (WT; GA-1981) and trf4Δ (GA-10632) cells were exponentially cultured in SC, then arrested in G1 with α-factor. G1-arrested cells were treated checked, and analyzed by CHEF gel and quantitation as in 5a. c Isogenic wild-type (WT; GA-741) and pol2-18 (GA-742) cells were exponentially cultured in SC at 25 °C, then arrested with α-factor. Cultures were shifted to 37 °C for 45 min, then treated with Zeocin CMB4563, or both, for 70 min prior to CHEF gel analysis and quantitation as above. d Loss of DNA ligase I (Cdc9) activity greatly enhances YCS. Isogenic wild-type (WT, GA-8709) and cdc9-1 (GA-8708) cells were grown exponentially in SC at 25 °C. Cells were arrested in G1 by α-factor for 2 h, then shifted to 37 °C for 45 min. Cells were treated with Zeocin, CMB4563, or both, as indicated for 60 min prior to CHEF gel analysis and quantitation as above. FACS analysis in Supplemental Fig. 5. Markers shown were run alongside the samples to determine the size of fragments produced in the cdc9-1 sample.

Fig. 6. Nuclear actin overexpression sensitizes cells to Zeocin, inducing DSBs.

a Wild-type GA-1981 cells were transformed with pCM190 vector (control), pCM190 carrying ACT1 (wild-type), or act1-nes (ACT1 bearing two mutated nuclear export signals), and were selected on SC-URA + 1 μg/ml doxycycline (Dox) to suppress pCM190 plasmid expression. The colonies transformed were diluted in a 5-fold series and spotted on SC-URA + /- 60 μg/ml Zeocin in the presence (+ Dox, OFF) or absence (no Dox, ON) of 1 μg/ml doxycycline. Growth was imaged after 3 days at 30 °C. b Chromatin fractionation shows that overexpressed act1-nes co-purifies with chromatin. Exponentially growing GA-1981 cells transformed as in (a) were subjected to chromatin fractionation. Overexpressed WT actin (Act1) was mostly found in the chromatin unbound fraction, while the majority of act1-nes co-purified with chromatin (as histone H4), indicating that act1-nes is retained in the nucleus and may be chromatin-bound. Fractionation controls and blots are in Source data files. c Actin mutants act1-S14C-nes, act1-AP-nes and Act1-nes (see text) are expressed like Act1-nes. The total extracted protein was subjected to Western blots with anti-actin and anti-H3. Original blots are provided in the Source Data file. d Strains expressing act1-S14C-nes, act1-AP-nes and Act1-nes hyperactivate Rad53 kinase on Zeocin vs vector control (wild-type). The total protein sample was subjected to Western blot probed with anti-Rad53 and anti-actin on the same blot (see Materials). Rad53 shows a significant shift due to phosphorylation by Mec1 and autophosphorylation. ns indicates a nonspecific cross-reactive band. Full blots are included in Source Data files. e The same strains bearing the indicated plasmids (see a and c) were exponentially cultured in SC-URA + 1 μg/ml Dox, then washed twice with SC-URA without Dox and cultured for 6 h without Dox to induce actin. Cells were treated with 0, 50, 150 μg/ml Zeocin for 80 min, and genomic DNA was subjected to CHEF gel and stained with HDGreen. Also tested was another actin mutant, act1-111, bearing the nes mutation. This actin mutant is polymerization-competent but can provoke YCS in the absence of CMB. Quantitation of YCS as in Fig. 1a and Supplementary Data 1.

Fig. 7. Enhanced Arp5 chromatin binding and DNA accessibility during YCS.

a Apn1 and Ogg1 co-precipitate with actin. Bead-bound bicyclic peptides specific for F-actin (A18), F/G actin (A15), or the control TATA2, were used to recover proteins from total yeast extracts. Proteins were visualized by silver staining, and prominent bands were identified by mass spectrometry from gel slices (see Methods, red = A15 and A18-pulldown, black = A15 only). The same fractions were analyzed by western blots for Apn1, Ogg1, Mcm2, and tubulin. Uncropped blots and mass spectroscopy results are in the Source data files. b Nuclear Apn1 levels drop slightly during YCS. APN1-GFP NUP49-RFP cells (GA-10504) cultured in SC were treated with DMSO ± 50 μg/ml Zeocin and 0.5 μM CMB4563 for 60 min. Spinning disc confocal images were captured of living cells in agarose plugs, and ImageJ quantified Apn1-GFP and Nup49-RFP intensities were plotted. Apn1-GFP was normalized to Nup49-RFP. Bar = 5 μm; n = cells imaged (n = 313, DMSO, and n = 763, Zeo + CMB). White bar = median; significance determined by Unpaired t test with Welch’s correction, two-tailed; p < 0.0001. c INO80 subunit Arp5, but not Apn1, is slightly enriched on chromatin during YCS. APN1-9Myc tagged cells exponentially cultured in SC were treated with DMSO ± 50 μg/ml Zeocin and 0.5 μM CMB4563 for 80 min, and subjected to chromatin fractionation^,. Total (T), soluble (S), and chromatin pellet (P) fractions were probed on western blots for Myc (9E10), Arp5, Orc2, and tubulin (see “Methods”). Full blots and quantitation in Source Data files. d P/T values for Apn1-Myc and Arp5 normalized to Orc2 are plotted, 1 = DMSO control. Anti-Orc2 cross-reacts with a 5kDa-smaller cytosolic protein (*). Uncropped blots are in Source data files. e Dam accessibility assay monitors the relative accessibility of GATC motifs to ectopically expressed methylase (sketch modified from). WT cells (GA-1981) carrying p415GAL (control) or p415GAL-Dam were treated with 50 μg/ml Zeocin, 0.5 μM CMB4563, or both for 80 min. Total genomic DNA was isolated, digested with DpnI, analyzed on a 1% agarose gel and stained by SYBR safe. DpnI-insensitive (intact genomic band) and DpnI-sensitive (below intact chromosomes) were quantified by ImageJ. Ratios from 5 biological replicas are plotted.

Fig. 8. Model of sequential BER of clustered damage and misregulation by nuclear actin.

We propose that the repair of clustered oxidization events on DNA becomes toxic in the presence of reagents that depolymerize actin in the cytoplasm, due to a miscoordination of the two-step BER process (see Discussion). The undisturbed step-wise repair of two closely positioned oxidation bases is shown on the left, with events that normally occur after the first lesion is repaired indicated in gray. Upon inhibition of TORC2, or disruption of cytoplasmic actin polymerization by other means, nuclear actin levels increase and provoke chromosome shattering in a manner dependent on Zeocin-induced damage and BER enzyme activity. In G1 phase cells, the increase in nuclear actin may activate Apn1/Apn2 directly or increase accessibility (see **) to drive premature processing of both lesions. In S-phase cells, nuclear actin may allow both glycosylases and AP endonucleases to prematurely process paired lesions. Unconstrained or enhanced processivity of DNA Pol δ in S phase, and Trf4 and DNA pol ε in both S and G1, contribute to DSB formation. The basic BER steps are original sketches based on recent reviews of BER pathways^–.