Yeast as a platform to dissect Magnaporthe oryaze poly(ADP-ribose) polymerase function and evaluate PARP inhibitors

12 Poly(ADP-ribose) polymerases (PARPs) regulate genome maintenance through NAD ⁺-13 dependent ADP-ribosylation, yet PARP function in fungi remains poorly defined. Here, we 14 reconstituted the activity of the Magnaporthe oryzae PARP1 homolog (MoPARP1) in 15 Saccharomyces cerevisiae , a genetically tractable organism that lacks endogenous 16 PARP enzymes. Galactose -induced expression of MoPARP1 reduced yeast growth, 17 whereas a catalytically inactive mutant showed no defect, indicating that the growth 18 phenotype depends on PARP catalytic activity. In a multidrug transporter -deficient 19 background, the PARP inhibitor 3-aminobenzamide and the clinically used PARP inhibitor 20 olaparib rescued the growth of MoPARP1-expressing strains, establishing a framework 21 for inhibitor testing in vivo. Finally, MoPARP1-GFP localized to the nucleus independent 22 of catalytic activity, supporting correct targeting in this heterologous system. Together, 23 these findings establish yeast as a platform to dissect fungal PARP biology and evaluate 24 chemical inhibition. 25 26

27 Poly(ADP-ribose) polymerases (PARPs) are a large and diverse family of ADP -28 ribosyltransferases (ARTs) found across eukaryotes. Using nicotinamide adenine 29 dinucleotide (NAD⁺) as a substrate, PARPs transfer ADP-ribose units onto target proteins 30 to generate either mono -ADP-ribosylation (MARylation) or poly(ADP -ribose) (PAR) 31 chains (1-4). In humans, at least 17 PARP family members have been identified. PARP1, 32 the most abundant and best -studied family member, catalyzes the majority of DNA 33 damage-induced PAR synthesis and regulates DNA repair, chromatin dynamics, 34 transcription, and cell death (5). Human PARP1 is a modular enzyme containing 35 regulatory motifs and accessory domains, including zinc finger DNA-binding domains, the 36 tryptophan-glycine-arginine (WGR) domain, and a BRCA1 C-terminal (BRCT) domain , 37 together with a conserved catalytic domain characterized by the histidine -tyrosine-38 glutamate (HYE) motif (3, 6). PAR signals are reversed primarily by poly(ADP -ribose) 39 glycohydrolase (PARG) and can be pharmacologically blocked using PARP inhibitors (6, 40 7). Disruption of PARP-mediated ADP-ribosylation is associated with neurodegeneration, 41 metabolic dysfunction, inflammatory diseases, and cancer (4, 8). Notably, the finding that 42 PARP1/2 inhibition can selectively kill BRCA-deficient tumor cells led to the development 43 of multiple FDA -approved PARP inhibitors (9-11). These advances underscore the 44 importance of defining PARP enzymology and the mechanisms by which ADP -45 ribosylation supports cellular homeostasis. 46 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint Despite extensive characterization of human PARPs, far less is known about PARP 47 function across other eukaryotic lineages. Plants encode multiple PARP homologs 48 implicated in DNA damage responses and genome maintenance, and they also contribute 49 to broader stress-response signaling (12-14). 50 Interest in PARP biology has recently expanded to filamentous fungi, where biological 51 roles have been difficult to dissect, in part because few systems enable robust in vivo 52 biochemical interrogation. In the rice blast fungus Magnaporthe oryzae , MoPARP1 -53 mediated PARylation of 14 -3-3 protein s, which regulate appressorium development, 54 mitogen-activated protein kinase (MAPK) signaling, and virulence, implicates ADP-55 ribosylation in infection -structure differentiation and host invasion (15). Similarly, in 56 Fusarium oxysporum f. sp. niveum, FonPARP1 has been shown to be an active nuclear 57 PARP whose activity is enhanced by kinase -dependent phosphorylation , and whose 58 substrate-specific PARylation of the protein disulfide isomerase FonPdi1 regulates protein 59 folding, endoplasmic reticulum homeostasis, and pathogenicity (16, 17). These studies 60 position fungal PARPs as virulence-linked regulators, supporting a reductionist approach 61 to evaluating PARP catalytic outputs and chemical sensitivity in a simplified cellular 62 background. 63 Heterologous gene expression in yeast provides a powerful approach to define PARP 64 function (18). Prior studies have characterized human and plant PARPs in 65 Saccharomyces cerevisiae (19-21). Because yeast lacks endogenous PARP genes and 66 does not synthesize PAR, it offers a genetically “clean” background in which all detected 67 ADP-ribosylation is derived from the introduced enzyme. This genetic simplicity, 68 combined with tractable manipulation and the absence of endogenous PARylation 69 machinery, makes yeast a uniquely useful platform for reconstructing and interrogating 70 PARP activities from diverse organisms. 71 In this study, we reconstitute and characterize the M. oryzae PARP1 homolog (MoPARP1) 72 in the PARP-free background of S. cerevisiae. We demonstrate that induced expression 73 of MoPARP1 in yeast leads to a strong, catalysis-dependent growth defect and localizes 74 to the nucleus. A catalytic -site mutant abolishes all enzymatic and phenotypic outputs, 75 confirming dependence on the conserved glutamate residue required for ADP -76 ribosyltransferase activity. Finally, we show that MoPARP1 is inhibited in vivo by 3 -77 aminobenzamide and olaparib, establishing a foundation for using yeast as a screening 78 platform for inhibitors targeting fungal PARPs. Together, our results provide the first 79 biochemical reconstruction of fungal PARP activity in yeast and introduce a versatile 80 system for functional analysis and discovery of antifungal inhibitors. 81 82

83 Expression of MoPARP1 reduces yeast growth in a catalytic-dependent manner 84 To assess whether expression of Magnaporthe oryzae PARP1 (MoPARP1) affects yeast 85 growth, we analyzed Saccharomyces cerevisiae BY4741 strains carrying an empty vector 86 (EV), wild -type MoPARP1, or a catalytically inactive mutant (MoPARP1 -E714A) under 87 repressing (glucose) and inducing (galactose) conditions. Human PARP1 (hsPARP1) was 88 included as a reference for PARP -dependent growth defects previously described in 89 yeast. 90 Under repressing conditions, all strains displayed comparable growth across serial 91 dilutions, indicating that basal expression did not measurably impact growth (Figure 1A). 92 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint Upon galactose induction, expression of MoPARP1 resulted in a marked reduction in 93 colony formation across the dilutions relative to EV controls. To verify that the observed 94 growth phenotypes were associated with induced PARP expression, we analyzed protein 95 levels by immunoblotting using an anti -MYC antibody. MoPARP1 and MoPARP1-E714A 96 were readily detected at the expected molecular weight in galactose -induced cultures, 97 whereas no signal was observed under repressing conditions or in EV controls (Figure 98 1B). In contrast, the expression of the catalytic mutant MoPARP1-E714A did not reduce 99 growth, with colony formation comparable to EV on both glucose and galactose media 100 (Figure 1A). These observations indicate that MoPARP1 -dependent growth inhibition in 101 yeast requires an intact catalytic residue. 102 Consistent with prior reports, the induction of h sPARP1 expression resulted in strong 103 growth inhibition on galactose plates (Figure 1 A). Notably, MoPARP1 expression 104 produced a qualitatively similar growth phenotype, supporting functional similarity 105 between fungal and human PARP proteins in this heterologous system. 106 To further characterize growth behavior, we monitored growth dynamics in liquid culture. 107 Under glucose conditions, all strains exhibited similar growth kinetics (Figure 2A, B), 108 consistent with the results of the spotting assay and confirming that the phenotype 109 depends on induced expression. In galactose conditions, strains expressing MoPARP1 110 or HsPARP1 exhibited a reproducible , pronounced growth delay and achieved lower 111 maximal optical densities compared to EV controls (Figure 2C, D). In contrast, MoPARP1-112 E714A displayed growth kinetics comparable to EV, consistent with its established effects 113 in yeast. Quantification of growth curves by area under the curve (AUC) analysis 114 supported these observations, with reduced AUC values for MoPARP1 - and HsPARP1-115 expressing strains relative to controls, while MoPARP1 -E714A showed no significant 116 difference (Figure 2C, D). Together, these data demonstrate that MoPARP1 impairs yeast 117 growth in a catalytic activity -dependent manner , producing a growth phenotype 118 comparable to that observed upon expression of HsPARP1. 119 PARP inhibitor treatment restores MoPARP1-dependent growth inhibition in yeast 120 To validate whether the growth inhibition associated with MoPARP1 expression is driven 121 by PARP enzymatic activity, we examined the effect of pharmacological PARP inhibition 122 in yeast. These experiments were performed in a BY4741 pdr5Δ background, which lacks 123 a major ATP -binding cassette multidrug efflux transporter and thereby enhances 124 intracellular accumulation of small molecules. 125 As observed in the wild -type background, induction of MoPARP1 expression in pdr5Δ 126 cells resulted in strong growth inhibition on galactose -containing media (Figure 3A, B). 127 The addition of the PARP inhibitor 3 -aminobenzamide (3 -AB; 5 mM), a classical 128 competitive inhibitor of PARP catalytic activity, partially rescued the growth defect of 129 MoPARP1-expressing strains, as evidenced by improved colony formation and increased 130 growth in liquid culture (Figure 3A,B). In contrast, the growth of the strain expressing the 131 catalytically inactive MoPARP1 -E714A mutant was unaffected by 3 -AB treatment, 132 consistent with the absence of PARP catalytic activity in this variant. 133 In line with its established behavior in yeast, the expression of HsPARP1 also caused 134 strong growth inhibition, which was similarly alleviated by 3 -AB treatment, serving as a 135 positive control for PARP inhibitor responsiveness (Figure 3A, B). Quantitative analysis 136 of growth curves using AUC measurements confirmed statistically significant restoration 137 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint of growth for MoPARP1- and HsPARP1-expressing strains upon 3-AB treatment, whereas 138 no significant change was observed for the catalytic mutant (Figure 3B, C). 139 To extend these findings using a mechanistically distinct inhibitor, we tested olaparib, a 140 clinically used HsPARP1 inhibitor that targets the conserved NAD⁺-binding pocket of the 141 PARP catalytic domain. Under galactose -inducing conditions, treatment with 25 µM 142 olaparib increased the growth of wild -type MoPARP1-expressing yeast in liquid culture 143 relative to untreated controls (Figure 4A, B). This rescue was supported by AUC -based 144 quantification, which showed a significant increase in AUC upon olaparib treatment ( p < 145 0.0001). Together with the 3 -AB results, these data indicate that MoPARP1 -dependent 146 growth inhibition in yeast is enzymatic in nature and can be mitigated by pharmacological 147 PARP inhibition. Collectively, these results demonstrate that MoPARP1 -dependent 148 growth inhibition in yeast can be chemically modulated by PARP inhibitors. The 149 concordant inhibitor responses further support functional conservation of key features of 150 the PARP catalytic mechanism and establish yeast as a tractable platform for assessing 151 pharmacological sensitivity of fungal PARP activity. 152 153 MoPARP1 localizes to the nucleus in yeast independent of its catalytic activity 154 To validate the yeast heterologous system and determine whether catalytic activity 155 influences subcellular localization, we examined the localization of MoPARP1 in S. 156 cerevisiae. Wild-type MoPARP1 and a catalytically inactive mutant, MoPARP1-EA, were 157 expressed as C -terminal GFP fusion proteins from a galactose -inducible vector in 158 BY4741 cells. Fluorescence microscopy showed that both MoPARP1 -GFP and 159 MoPARP1-EA-GFP localized exclusively to the nucleus, as indicated by the overlap with 160 Hoechst staining and no detectable cytoplasmic signal (Figure 5). These results confirm 161 correct nuclear targeting of MoPARP1 in yeast and indicate that differences in growth 162 phenotypes between the wild -type and catalytic mutant proteins are not due to altered 163 subcellular localization. 164 165 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint 166 Figure 1. Galactose -inducible expression of PARP1 fusion proteins in yeast and 167 verification of expression. (A) BY4741 yeast strains expressing the empty vector (EV) 168 or indicated PARP1-myc fusion constructs were grown overnight in repressing medium 169 (2% glucose). Cultures were normalized to an OD600 of 1.0, serially diluted 10-fold, and 170 spotted onto plates containing either repressing medium (2% glucose) or inducing 171 medium (2% galactose). Plates were imaged after 2 days of incubation at 30 °C. (B) 172 Induced cultures from (A) were harvested at equal cell equivalents, lysed, and analyzed 173 by SDS -PAGE followed by immunoblotting with an anti -MYC antibody to confirm 174 expression at the expected molecular weights (MoPARP1, 87 kDa; HsPARP1, 110 kDa). 175 176 177 178 179 180 181 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint 182 183 184 Figure 2. MoPARP1 inhibits yeast growth in a catalytic -dependent manner. (A) 185 OD₆₀₀ growth curves of S. cerevisiae BY4741 expressing empty vector (EV), MoPARP1, 186 catalytic mutant MoPARP1 -E714A, or human PARP1 (HsPARP1) under repressing 187 conditions (glucose). (B) Area under the curve (AUC) analysis of (A) shows no significant 188 differences. (C) Under inducing conditions (galactose), expression of MoPARP1 and 189 HsPARP1 reduced yeast growth compared with EV, whereas MoPARP1-E714A showed 190 no significant effect. (D) AUC quantification of (C). MoPARP1 and HsPARP1 significantly 191 reduced growth relative to EV, while MoPARP1 -E714A did not. Data are presented as 192 mean ± SD; statistical significance was determined by one -way ANOVA with Dunnett’s 193 multiple comparisons test; ****, P < 0.0001; ns, not significant. 194 195 196 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint 197 Figure 3. PARP inhibition rescues MoPARP1 -mediated growth inhibition under 198 inducing conditions. (A) Serial dilution spot assays of BY4741 pdr5Δ expressing empty 199 vector (EV), MoPARP1, catalytic mutant MoPARP1 -E714A, or HsPARP1. Cells were 200 grown on glucose (repressing) or galactose (inducing) media in the presence of 5 mM 3-201 aminobenzamide (3-AB). (B) Liquid growth curves (OD₆₀₀) under inducing conditions ± 5 202 mM 3 -AB. (C) Area under the curve (AUC) quantification of (B). 3 -AB significantly 203 increased growth of MoPARP1 -expressing cells but had no significant effect on 204 MoPARP1-E714A. Data are presented as mean ± SD. Statistical significance was 205 determined by two-way ANOVA with Šídák’s multiple comparisons test; ****, P < 0.0001; 206 ns, not significant. 207 208 209 210 211 212 213 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint 214 215 216 Figure 4 . Effect of the PARP inhibitor olaparib on MoPARP1 -dependent growth 217 under inducing conditions. Growth of BY4741 pdr5Δ cells expressing wild -type 218 MoPARP1 was measured in liquid culture under galactose -inducing conditions in the 219 absence or presence of olaparib (25 µM). Growth was monitored by OD₆₀₀ measurements 220 over time and quantified by area under the curve (AUC) analysis. Data are presented as 221 mean ± SD from three biological replicates. Statistical significance was determined using 222 an unpaired two-tailed t-test with Welch’s correction; ****, P < 0.0001; ns, not significant. 223 224 225 226 227 228 229 230 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint 231 Figure 5. Subcellular localization of galactose -inducible MoPARP1-eGFP fusion 232 proteins in BY4741 yeast cells. BY4741 cells expressing galactose -inducible 233 MoPARP1-eGFP fusion constructs were grown under inducing conditions (galactose) and 234 analyzed by fluorescence microscopy. eGFP fluorescence was used to visualize the 235 subcellular distribution of MoPARP1. Nuclei were stained with Hoechst to serve as a 236 nuclear marker and assess co-localization with MoPARP1-eGFP. Boxed regions indicate 237 areas shown at higher magnification in the inset panels to highlight subcellular 238 localization. Representative fluorescence and corresponding brightfield images are 239 shown. Scale bars = 5 μm 240 241 242 243 244 245 246 247 248 249 250 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint

251 Yeast heterologous expression systems provide a powerful way to dissect protein function 252 in a simplified eukaryotic context, particularly for enzymatic activities that are absent from 253 S. cerevisiae and therefore lack confounding endogenous regulation. Because yeast 254 lacks canonical poly(ADP -ribose) polymerases, it provides an essentially background -255 free platform for isolating PARP -dependent effects and directly connecting catalytic 256 activity to cellular phenotypes (18, 19, 22, 23). 257 PARylation is a highly dynamic post -translational modification that can rapidly reshape 258 protein interactions, chromatin organization, and stress -responsive signaling. Because 259 PARP activity is tightly coupled to NAD ⁺ utilization and generates an amplifying 260 biochemical output, even modest deregulation of PARylation can impose substantial 261 fitness costs. In this context, yeast offers a controlled eukaryotic system in which PARP 262 activity can be examined independently of organism-specific developmental programs or 263 signaling networks. Here, we leverage this system to evaluate the cellular consequences 264 of induced M. oryzae PARP1 expression, focusing on growth behavior, subcellular 265 localization, and pharmacological sensitivity. 266 Using this approach, expression of wild -type MoPARP1 in yeast wielded a pronounced 267 fitness cost, evident in both spot dilution assays and liquid growth curves, whereas growth 268 under repressing conditions remained unaffected. These observations indicate that 269 MoPARP1 is enzymatically active in vivo and that its activity alone is sufficient to disrupt 270 cellular growth in a heterologous eukaryotic system. Comparable growth inhibition 271 phenotypes have been reported upon expression of plant and mammalian PARPs in 272 yeast, supporting the idea that deregulated PARP activity is intrinsically deleterious when 273 uncoupled from native regulatory constraints (19, 24, 25). However, it remains unknown 274 which downstream processes are affected by MoPARP1 activity. Given the link between 275 PARP activity and NAD⁺ consumption, future work should investigate whether the growth 276 defect results from NAD ⁺ depletion/metabolic stress, and whether it activates yeast 277 stress-response pathways. 278 The nuclear localization of MoPARP1 in yeast further supports its functional relevance in 279 this system and is consistent with the established chromatin -associated roles of PARP 280 proteins across eukaryotes. MoPARP1 accumulated predominantly in the nucleus under 281 inducing conditions, indicating that nuclear targeting is preserved in the heterologous 282 background. PARP localization has also been examined in M. oryzae, where PARP1-GFP 283 fluorescence was readily detected in the nuclei of hyphal cells, while nuclear localization 284 in three-celled conidia was not clearly observed under the conditions tested (15). The 285 conserved nuclear enrichment observed in hyphae and in yeast supports the view that 286 nuclear targeting is an intrinsic property of MoPARP1 and reinforces the relevance of 287 yeast as a system for assessing MoPARP1 localization and function. It remains unclear 288 whether nuclear localization is required for MoPARP1 -mediated toxicity and whether 289 specific chromatin-associated interactions underline the observed growth defects. 290 Chemical inhibition experiments further support the enzymatic basis of the MoPARP1 -291 dependent growth phenotype. Treatment with the 3 -AB and the human PARP inhibitor 292 olaparib alleviated MoPARP1 -dependent growth inhibition in a multidrug transporter -293 deficient (pdr5Δ) background, consistent with suppression of PARP catalytic activity. 3-294 AB is a classical competitive PARP inhibitor that binds the conserved nicotinamide -295 binding pocket within the NAD ⁺ site, thereby blocking ADP-ribosyl transfer. The ability to 296 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint chemically modulate MoPARP1 activity in yeast further highlights the utility of this system 297 for probing PARP enzymatic function. These inhibitor rescue experiments raise questions 298 about which features of the MoPARP1 catalytic domain govern inhibitor sensitivity and 299 whether other clinically relevant PARP inhibitors (PARPis) show similar activity against 300 fungal PARPs. 301 PARPis have become an important class of targeted therapeutics, particularly in ovarian 302 cancer, and several agents, including olaparib, niraparib, and rucaparib, are approved by 303 the U.S. FDA for maintenance therapy (9). Olaparib was the first PARPi approved as first-304 line maintenance monotherapy, based on the phase III trial. Mechanistically, olaparib 305 binds to the conserved NAD ⁺-binding pocket of PARP enzymes, inhibiting catalytic 306 PARylation and also stabilizing PARP-DNA complexes, thereby promoting DNA damage 307 repair in cells with PARP deficiency (7). Because olaparib is well studied clinically and 308 mechanistically, it provides a useful pharmacological probe for assessing PARP -309 dependent phenotypes. In the context of this study, the responsiveness of MoPARP1 to 310 olaparib further supports conservation of key catalytic features and validates the use of 311 yeast as a platform for probing fungal PARP activity. 312 More broadly, yeast has been widely used as a platform to evaluate PARP inhibitor 313 sensitivity, define structure-function relationships, and prioritize small molecules based on 314 phenotypic rescue (19, 23, 26) . In this context, MoPARP1 -dependent growth inhibition 315 provides a clear and tractable readout that can be leveraged to assess inhibitor 316 responsiveness and guide downstream validation in M. oryzae . While compounds 317 identified in yeast would require further characterization for specificity, target 318 engagement, and efficacy in the native pathogen, this approach establishes a foundation 319 for developing chemical probes to interrogate fungal PARP function and explore PARP 320 inhibition as a means to modulate fungal stress responses relevant to pathogenic fitness. 321 322

323 Cloning and vectors 324 The coding sequence of Magnaporthe oryzae PARP1 (MoPARP1) was amplified from M. 325 oryzae cDNA using Q5 High -Fidelity DNA Polymerase (New England Biolabs). PCR 326 reactions were set up in 25 µL volumes containing 1× Q5 reaction buffer, 200 µM of each 327 dNTP, 0.5 µM of each forward and reverse primer, and approximately 50 ng of template 328 cDNA. A typical cycling program consisted of an initial denaturation at 98 °C for 30 s, 329 followed by 30 cycles of 98 °C for 10 s, 55-60 °C for 20 s, and 72 °C for 1-2 min, depending 330 on amplicon length, with a final extension at 72 °C for 5 min. The catalytic mutant 331 MoPARP1-E714A was generated by site-directed mutagenesis with Q5 using overlapping 332 primers carrying the desired glutamate -to-alanine substitution at position 714; after 333 amplification, parental template DNA was digested with DpnI at 37 °C for 1 -2 h. Human 334 PARP1 coding sequence was obtained from Addgene plasmids ( HsPARP1, plasmid 335 #111574) and amplified using Q5 with primer pairs designed to introduce overlaps 336 compatible with Gibson assembly into the pESC-Leu vector (Figure S1). All PCR products 337 (MoPARP1, MoPARP1-E714A, and HsPARP1) were purified with a PCR cleanup kit and 338 inserted into the GAL1-driven pESC-Leu backbone using NEBuilder HiFi DNA Assembly 339 Master Mix (New England Biolabs) following the manufacturer’s instructions. For 340 subcellular localization, the MoPARP1 coding sequence was amplified using Q5 and 341 cloned in frame with GFP into the URA3-selectable pD-eGFP vector via Gibson assembly. 342 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint All constructs were confirmed by restriction digestion and Sanger sequencing to verify the 343 correct insert sequence and the presence of the E714A mutation. 344 345 Bacterial transformation and plasmid preparation 346 Recombinant pESC -Leu and pD -eGFP plasmids were propagated in Escherichia coli 347 Top10. Chemically competent Top10 cells were transformed by heat shock at 42 °C for 348 60 seconds, recovered in SOC medium at 37 °C for 1 hour, and then plated on LB agar 349 containing the appropriate antibiotic. Single colonies were grown overnight at 37 °C in 3-350 5 mL LB medium with antibiotics, and plasmid DNA was purified using spin miniprep kits. 351 Plasmids were screened by diagnostic restriction digestion and then sequenced to 352 confirm the identity and integrity of MoPARP1, MoPARP1-E714A, and HsPARP1 inserts 353 before yeast transformation. 354 355 Yeast strains, media, and transformation 356 All yeast experiments were performed in the Saccharomyces cerevisiae strain BY4741 357 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and in an isogenic BY4741 -pdr5Δ mutant 358 (YOR153W; Horizon Discovery), which was used to enhance intracellular accumulation 359 of small -molecule inhibitors in uptake -sensitive assays. For selection of pESC -Leu 360 constructs, strains were maintained on synthetic dropout medium lacking leucine (S D-361 Leu), whereas selection of pD-GFP constructs for localization was carried out on synthetic 362 complete medium lacking uracil (S D-Ura). For repression of GAL1 -driven PARP 363 expression, cells were cultured in S D medium supplemented with 2% (w/v) glucose as 364 the carbon source. For induction of PARP expression, glucose was replaced with 2% 365 (w/v) galactose. Cultures were routinely grown at 30 °C with shaking at 200 rpm. Yeast 366 transformations were performed using the lithium acetate/polyethylene glycol method. 367 Briefly, BY4741 or BY4741 -pdr5Δ cultures were grown to mid -log phase (OD ₆₀₀ 368 approximately 0.4 -0.6), harvested by centrifugation, washed with sterile water, and 369 resuspended in 100 mM lithium acetate. Aliquots of competent cells were mixed with 370 plasmid DNA (typically 200–500 ng), boiled salmon sperm carrier DNA, lithium acetate , 371 and PEG 3350, incubated at 30 °C for 30 min, and subjected to a heat shock at 42 °C for 372 15 min. Transformed cells were recovered briefly in nonselective medium when needed 373 and plated on S D-Leu or S D-Ura agar for selection. Colonies were re -streaked and 374 verified by colony PCR where necessary. For all experiments, BY4741 WT and BY4741-375 pdr5Δ were transformed with the empty vector and with each PARP construct. 376 377 Spotting assays on glucose and galactose 378 The effect of MoPARP1, MoPARP1-E714A, and HsPARP1 expression on yeast growth 379 was assessed by spotting assays under repressing (glucose) and inducing (galactose) 380 conditions. Single colonies from S D-Leu plates were inoculated into 3 -5 mL S D-Leu 381 containing 2% glucose and cultured overnight at 30 °C with shaking. Overnight cultures 382 were harvested by centrifugation, washed twice with sterile water to remove residual 383 glucose, and resuspended in sterile water. Cell density was adjusted to OD₆₀₀ = 0.5, and 384 a series of ten-fold serial dilutions was prepared. Aliquots of 3 µL from each dilution were 385 spotted onto SD-Leu plates containing either 2% glucose or 2% galactose. Plates were 386 incubated at 30 °C for 48-72 h, until colonies were clearly visible, and then photographed. 387 Growth patterns were compared between glucose and galactose for each strain to 388 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint determine whether induction of PARP expression resulted in growth inhibition. Spotting 389 assays were performed in both BY4741 and BY4741-pdr5Δ backgrounds using the same 390 constructs and empty vector controls. 391 392 Cell lysis and immunoblotting 393 For yeast cell lysis, 1 mL of galactose -induced cultures was collected , and cells were 394 harvested by centrifugation. Pellets were resuspended in 100 µL of ice-cold lysis solution 395 (4% v/v 10 N NaOH, 0.5% v/v β-mercaptoethanol) and incubated on ice for 30 min. 396 Lysates were adjusted to pH 9 –10 with HCl, and 2× Laemmli sample buffer was added. 397 Proteins were separated by SDS -PAGE and transferred to polyvinylidene difluoride 398 (PVDF) membranes. MoPARP1 expression was detected by immunoblotting using anti -399 Myc antibody (1:15,000; clone 9E10, SC-40; Santa Cruz Biotechnology). 400 401 Liquid growth curve analysis 402 To quantify the effect of PARP expression on yeast growth, liquid growth curves were 403 generated in microplate format. Overnight cultures grown in SD -Leu containing glucose 404 were harvested, washed twice with sterile water, and diluted into SD -Leu containing 2% 405 galactose to an initial OD ₆₀₀ of 0.1. For microplate assays, 100 µL of each culture was 406 dispensed into wells of a sterile 96 -well plate in triplicate for each strain and condition. 407 Plates were sealed with a breathable membrane and incubated at 30 °C in a microplate 408 reader with orbital shaking. Optical density at 600 nm was measured every 15 minutes 409 for 36 hours. Growth curves were generated by plotting OD ₆₀₀ versus time. Quantitative 410 analysis of growth curves and area under the curve (AUC) measurements was performed 411 using GraphPad Prism (GraphPad Software, Boston, MA, USA). 412 413 Inhibitor treatment 414 Pharmacological inhibition of PARP activity in vivo was examined using 3 -415 aminobenzamide (3 -AB) and olaparib. A concentrated stock solution of 3 -AB was 416 prepared fresh in sterile water, typically at 25 mM. Olaparib was prepared as a 417 concentrated stock solution in DMSO and added to cultures at a final concentration of 25 418 µM. For inhibitor assays, BY4741 -pdr5Δ strains carrying pESC -Leu empty vector, 419 MoPARP1-WT, MoPARP1-E714A, or HsPARP1 were grown overnight in S D-Leu with 420 glucose, washed, and resuspended in SD-Leu with galactose to induce PARP expression. 421 For 3-AB treatment, liquid cultures were supplemented with 3-AB to a final concentration 422 of 5 mM, with equivalent volumes of solvent added to control cultures. For plate -based 423 assays, serial dilution spotting was performed on SD-Leu agar plates containing 5 mM 3-424 AB. For liquid growth assays, cultures were grown in SD-Leu + galactose in the presence 425 or absence of the inhibitor, as described above, and the OD₆₀₀ was monitored over time. 426 For olaparib treatment, assays were performed exclusively in liquid culture under inducing 427 conditions. Growth was monitored by OD₆₀₀ over time to generate growth curves, and the 428 AUC for each replicate was calculated in GraphPad Prism (GraphPad Software, Boston, 429 MA, USA) and used for quantitative comparison between inhibitor-treated and untreated 430 cultures within each construct. The extent to which olaparib alleviated PARP -dependent 431 growth inhibition relative to untreated controls was used as a measure of inhibitor 432 sensitivity in the yeast system. 433 434 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.29.696956doi: bioRxiv preprint Fluorescence microscopy for MoPARP1-GFP localization 435 Subcellular localization of MoPARP1 was examined in BY4741 cells expressing 436 MoPARP1-GFP from the URA3 -selectable pD-GFP vector. Transformants were grown 437 overnight in S D-Ura medium containing 2% glucose, then shifted to S D-Ura medium 438 containing 2% galactose to induce expression for 6 h at 30 °C. Cells were collected by 439 gentle centrifugation, washed once with 1× phosphate -buffered saline (PBS), and fixed 440 with 4% paraformaldehyde. Following fixation, cells were washed twice with 1× PBS and 441 resuspended in a small volume of PBS. For nuclear staining, Hoechst (Sigma) was added 442 to the cell suspension to a final concentration of approximately 1 µg/mL and incubated 443 briefly before mounting on microscope slides. Cells were imaged using a fluorescence 444 microscope (Zeiss Axio Observer 7), and representative images were captured. Images 445 were processed using Fiji (ImageJ distribution) (27). Images were created in 446 https://BioRender.com. 447 448 Statistical analysis 449 All experiments were performed with at least three independent biological replicates. 450 Growth curve data were analyzed using GraphPad Prism (GraphPad Software, Boston, 451 MA, USA). Differences in growth between strains and conditions were evaluated using 452 two-way ANOVA followed by Dunnett’s and Šídák’s multiple-comparisons test. Area under 453 the curve values were calculated using the trapezoidal method and are reported as mean 454 ± SD. P values < 0.05 were considered statistically significant. 455 456 Author Contribution 457 J.F. conceived the study and designed the experiments. J.F., N.P., and R.E.K, conducted 458 the experiments. J.F., N.P., and R.E.K. wrote and revised the final manuscript. 459 460

461 We thank colleagues whose work could not be cited due to space and scope limitations. 462 We also thank the Fernandez laboratory for constructive feedback. 463 464 Conflict of Interest 465 The authors declare no conflict of interest. 466 Funding Information 467 This work received no specific grant from any funding agency . R.E.K. was supported by 468 the University of Florida Office of Research through the Research Opportunity Seed Fund 469 (ROSF). 470 471

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