DNA Damage Research Articles

Temporal regulation of gene expression through integration of p53 dynamics and modifications.

The master regulator of the DNA damage response, the transcription factor p53, orchestrates multiple downstream responses and coordinates repair processes. In response to double-strand DNA breaks, p53 exhibits pulses of expression, but how it achieves temporal coordination of downstream responses remains unclear. Here, we show that p53's posttranslational modification state is altered between its first and second pulses of expression. We show that acetylations at two sites, K373 and K382, were reduced in the second pulse, and these acetylations differentially affected p53 target genes, resulting in changes in gene expression programs over time. This interplay between dynamics and modification may offer a strategy for cellular hubs like p53 to temporally organize multiple processes in individual cells.

Protective Effects of Anthocyanin and α-Tocopherol Against Titanium Dioxide Nanoparticle-Induced DNA Damage in Human Sperm Cells

Protective Effects of Anthocyanin and α-Tocopherol Against Titanium Dioxide Nanoparticle-Induced DNA Damage in Human Sperm Cells

Arterial hypertension and significant DNA damage - from cell lines to patients

Arterial hypertension and significant DNA damage - from cell lines to patients

Clinical relevance of protein-truncating variants of germline DNA repair genes in prostate cancer

BackgroundInterpreting genetic variants remains a challenge in prostate cancer (PCa). Although many annotation tools are available for prioritizing causal variants, the clinical relevance of these variants is rarely studied.MethodsWe collected a cohort study that included 274 PCa patients from June 2017 to December 2020 and sequenced 19 DNA damage repair (DDR) genes in these patients and explored the clinical consequence of these different approaches. We also examined all-cause and PCa-specific survival in DDR gene mutation carriers compared to non-carriers after androgen receptor (AR)-directed therapy.ResultsWe identified 13 variants from 19 DDR genes in a total of 14 (5.1%) patients who had at least one presumed pathogenic mutation using different annotation methods. Four variants were annotated as pathogenic, 11 variants were predicted as protein-truncating variants (PTVs), four variants received proxy-deleterious (Combined Annotation-Dependent Depletion scores of > 30), and only one variant was identified as a pathogenic variant or as having a functional effect by all three methods. PCa patients with PTVs were significantly associated with early onset, high cancer stage, and a worse response to AR-directed treatment. However, patients carrying a proxy-deleterious variant were only associated with a higher T (tumor) stage and N (node) stage than those without such a variant, but not associated with other clinical characteristics. In patients treated with AR-directed therapy, patients with a PTV showed an increased risk of all-cause death (adjusted hazard ratio (aHR) = 3.51, 95% confidence interval (CI): 1.06 ~ 11.56) and PCa-specific death (aHR = 4.49, 95% CI: 1.87 ~ 10.77) compared to non-PTV carriers after adjustment. We were unable to examine gene-specific risks due to the small number of patients.ConclusionsPTVs may assist in guiding treatment and early screening in PCa, while population-specific data for pathogenic variants are still being amassed.

Germline DNA damage repair variants and prognosis of patients with high-risk or metastatic prostate cancer.

Deleterious germline variants in certain DNA repair genes are risk factors for developing aggressive prostate cancer. The objective was to quantify their prognostic impact after prostate cancer diagnosis. Men with prostate cancer, predominantly of European ancestry, were included from four cohorts with long-term follow-up. Pathogenic or likely pathogenic germline variants in 26 DNA repair genes were assessed in relation to metastasis-free survival in high-risk, localized prostate cancer and to overall survival in metastatic castration-sensitive prostate cancer (mCSPC) and metastatic castration-resistant prostate cancer (mCRPC). Among 3,525 patients initially diagnosed with non-metastatic prostate cancer, 2,594 had high-risk localized prostate cancer, 429 mCSPC, and 502 mCRPC at inclusion. BRCA2 variant carriers did not have worse metastasis-free survival in high-risk localized prostate cancer (hazard ratio [HR] 1.01, 95% confidence interval [CI] 0.69, 1.46) or overall survival in mCSPC (HR 0.46, 95% CI 0.14, 1.45) or mCRPC (HR 0.60, 95% CI 0.31, 1.17) compared to non-carriers of DNA repair variants. Among 868 additional patients with de novo metastatic (M1) prostate cancer, BRCA2 variant carriers tended to have worse overall survival (HR 1.59, 95% CI 1.01, 2.51). BRCA2 prognostic associations were not explained by radiation, PARP inhibitor, or platinum therapy. Results for other genes were limited in precision because variants were less common. Among patients with high-risk or metastatic prostate cancer who were initially diagnosed with and treated for non-metastatic tumors, germline DNA repair variants in BRCA2 do not confer a substantially worse prognosis.

Uncoupling of mTORC1 from E2F activity maintains DNA damage and senescence

DNA damage is a primary trigger for cellular senescence, which in turn causes organismal aging and is a promising target of anti-aging therapies. Most DNA damage occurs when DNA is fragile during DNA replication in S phase, but senescent cells maintain DNA damage long-after DNA replication has stopped. How senescent cells induce DNA damage and why senescent cells fail to repair damaged DNA remain open questions. Here, we combine reversible expression of the senescence-inducing CDK4/6 inhibitory protein p16INK4 (p16) with live single-cell analysis and show that sustained mTORC1 signaling triggers senescence in non-proliferating cells by increasing transcriptional DNA damage and inflammation signaling that persists after p16 is degraded. Strikingly, we show that activation of E2F transcriptional program, which is regulated by CDK4/6 activity and promotes expression of DNA repair proteins, repairs transcriptionally damaged DNA without requiring DNA replication. Together, our study suggests that senescence can be maintained by ongoing mTORC1-induced transcriptional DNA damage that cannot be sufficiently repaired without induction of protective E2F target genes.

IKKα affects the susceptibility of primary human osteoarthritis chondrocytes to oxidative stress-induced DNA damage by tuning autophagy

The functional derangement affecting human chondrocytes during osteoarthritis (OA) onset and progression is sustained by the failure of major homeostatic mechanisms. This makes them more susceptible to oxidative stress (OS), which can induce DNA damage responses and exacerbate stress-induced senescence. The knockdown (KD) of IκB kinase α (IKKα), a dispensable protein in healthy articular cartilage physiology, was shown to increase the survival and replication potential of human primary OA chondrocytes. Our recent findings showed that the DNA Mismatch Repair pathway only partially accounts for the reduced susceptibility to OS of IKKαKD cells. Here we therefore investigated other ROS-mediated DNA damage and repair mechanisms. We exposed IKKαWT and IKKαKD chondrocytes to sub-cytotoxic hydrogen peroxide and evaluated the occurrence of double-strand breaks (DSB), 8-oxo-2′-deoxyguanosine (8-oxo-dG) and telomere shortening. ROS exposure was able to significantly increase the number of γH2AX foci (directly related to the number of DSB) in both cell types, but IKKα deficient cells undergoing cell division were able to better recover compared to their IKKα proficient counterpart. 8-oxo-dG signal proved to be the highest DNA damage signal among those investigated, located in the mitochondria and with a slightly higher intensity in IKKα proficient cells immediately after OS exposure. Furthermore, ROS significantly reduced telomere length both in IKKαWT and IKKαKD, with the former showing more pervasive effects, especially in dividing cells. Assessment of the HIF-1α>Beclin-1>LC3B axis after recovery from OS showed that IKKα deficient cells exhibited a more efficient autophagic machinery that allowed them to better cope with oxidative stress, possibly through the turnover of damaged mitochondria. Higher Beclin-1 levels likely helped in rescuing dividing cells (identified by coupled cell cycle analysis) because of Beclin-1's involvement in both autophagy and mitotic spindle organization. Therefore, our data further confirm the higher capacity of IKKαKD chondrocytes to cope with oxidative stress-induced DNA damage.

TFAM as a sensor of UVC-induced mitochondrial DNA damage.

Mitochondria lack nucleotide excision DNA repair; however, mitochondrial DNA (mtDNA) is resistant to mutation accumulation following DNA damage. These observations suggest additional damage sensing or protection mechanisms. Transcription Factor A, Mitochondrial (TFAM) compacts mtDNA into nucleoids. As such, TFAM has emerged as a candidate for protecting DNA or sensing damage. To examine these possibilities, we used live-cell imaging, cell-based assays, atomic force microscopy, and high-throughput protein-DNA binding assays to characterize the binding properties of TFAM to UVC-irradiated DNA and cellular consequences of UVC irradiation. Our data indicate an increase in mtDNA degradation and turnover, without a loss in mitochondrial membrane potential that might trigger mitophagy. We identified a reduction in sequence specificity of TFAM associated with UVC irradiation and a redistribution of TFAM binding throughout the mitochondrial genome. Our AFM data show increased compaction of DNA by TFAM in the presence of damage. Despite the TFAM-mediated compaction of mtDNA, we do not observe any protective effect on DNA damage accumulation in cells or in vitro . Taken together, these studies indicate that UVC-induced DNA damage promotes compaction by TFAM, suggesting that TFAM may act as a damage sensor, sequestering damaged genomes to prevent mutagenesis by direct removal or suppression of replication.

ATR promotes mTORC1 activity via de novo cholesterol synthesis.

DNA damage and cellular metabolism exhibit a complex interplay characterized by bidirectional feedback mechanisms. Key mediators of the DNA damage response and cellular metabolic regulation include Ataxia Telangiectasia and Rad3-related protein (ATR) and the mechanistic Target of Rapamycin Complex 1 (mTORC1), respectively. Previous studies have established ATR as a regulatory upstream factor of mTORC1 during replication stress; however, the precise mechanisms by which mTORC1 is activated in this context remain poorly defined. Additionally, the activity of this signaling axis in unperturbed cells has not been extensively investigated. Here, we demonstrate that ATR promotes mTORC1 activity across various cellular models under basal conditions. This effect is particularly enhanced in cells following the loss of p16, which we have previously associated with hyperactivation of mTORC1 signaling and here found have increased ATR activity. Mechanistically, we found that ATR promotes de novo cholesterol synthesis and mTORC1 activation through the upregulation of lanosterol synthase (LSS), independently of both CHK1 and the TSC complex. Furthermore, the attenuation of mTORC1 activity resulting from ATR inhibition was rescued by supplementation with lanosterol or cholesterol in multiple cellular contexts. This restoration corresponded with enhanced localization of mTOR to the lysosome. Collectively, our findings demonstrate a novel connection linking ATR and mTORC1 signaling through the modulation of cholesterol metabolism.

Immunomodulatory effects of trastuzumab deruxtecan through the cGAS-STING pathway in gastric cancer cells

Although the efficacy of trastuzumab deruxtecan (T-DXd) against HER2-positive gastric cancers (GCs) has driven its clinical application, the precise mechanisms governing its immunomodulatory role remain unclear. In this study, we examined the immune-related mechanisms of action of T-DXd in GC cells. T-DXd exhibited potent antitumor effects in GC cells across diverse HER2 expression levels by inducing DNA damage and apoptosis. Activation of the DNA damage response by T-DXd led to increased PD-L1 expression. RNA-Seq analysis revealed that T-DXd modulated immune-related pathways, resulting in the upregulation of genes associated with inflammation and IFN signaling. Importantly, T-DXd activated the cGAS-STING pathway, inducing an IFN-I response in HER2-positive GC cells. Furthermore, T-DXd activated dendritic cells via the cancer cell-intrinsic cGAS-STING-IFN axis and enhanced PBMC-mediated tumor cell killing by activating CD8+ T cells. These findings provide valuable insights into the role of the cytosolic DNA sensing pathway in the action of T-DXd and offer a compelling rationale for combining T-DXd with immune checkpoint blockade therapies in GC treatment.

Interpreting the biological effects of protons as a function of physical quantity: linear energy transfer or microdosimetric lineal energy spectrum?

The choice of appropriate physical quantities to characterize the biological effects of ionizing radiation has evolved over time coupled with advances in scientific understanding. The basic hypothesis in radiation dosimetry is that the energy deposited by ionizing radiation initiates all the consequences of exposure in a biological sample (e.g., DNA damage, reproductive cell death). Physical quantities defined to characterize energy deposition have included dose, a measure of the mean energy imparted per unit mass of the target, and linear energy transfer (LET), a measure of the mean energy deposition per unit distance that charged particles traverse in a medium. The primary advantage of using the “dose and LET” physical system is its relative simplicity, especially for presenting and recording results. Inclusion of additional information such as the energy spectrum of charged particles renders this approach adequate to describe the biological effects of large dose levels from homogeneous sources. The primary disadvantage of this system is that it does not provide a unique description of the stochastic nature of radiation interactions. We and others have used dose-averaged LET (LETd) as a correlative physical quantity to the relative biological effectiveness (RBE) of proton beams. This approach is based on established experimental findings that proton RBE increases with LETd. However, this approach might not be applicable to intensity-modulated proton therapy or other applications in which the proton energy spectrum is highly heterogeneous. In the current study, we irradiated cancer cells with scanning proton beams with identical LETd (3.4 keV/µm) but arising from two different proton energy/LET spectra (a narrow spectrum in group 1 and a widespread heterogeneous spectrum in group 2). Clonogenic survival after irradiation revealed significant differences in RBE at any cell surviving fraction: e.g., at a surviving fraction of 0.1, the RBE was 0.97 ± 0.03 in group 1 and 1.16 ± 0.04 in group 2 (p≤0.01), validating our hypothesis that LETd alone may not adequately indicate proton RBE. Further analysis showed that microdosimetric spectrum (the probability density function of the stochastic physical quantity lineal energy y) was helpful for interpreting observed differences in biological effects. However, more accurate use of microdosimetric spectrum to quantify RBE requires a cell line–specific mechanistic model.

WRAD core perturbation impairs DNA replication fidelity promoting immunoediting in pancreatic cancer.

It is unclear how cells counteract the potentially harmful effects of uncoordinated DNA replication in the context of oncogenic stress. Here, we identify the WRAD (WDR5/RBBP5/ASH2L/DPY30) core as a modulator of DNA replication in pancreatic ductal adenocarcinoma (PDAC) models. Molecular analyses demonstrated that the WRAD core interacts with the replisome complex, with disruption of DPY30 resulting in DNA re-replication, DNA damage, and chromosomal instability (CIN) without affecting cancer cell proliferation. Consequently, in immunocompetent models, DPY30 loss induced T cell infiltration and immune-mediated clearance of highly proliferating cancer cells with complex karyotypes, thus improving anti-tumor efficacy upon anti-PD-1 treatment. In PDAC patients, DPY30 expression was associated with high tumor grade, worse prognosis, and limited response to immune checkpoint blockade. Together, our findings indicate that the WRAD core sustains genome stability and suggest that low intratumor DPY30 levels may identify PDAC patients who will benefit from immune checkpoint inhibitors.

Genetic iron overload aggravates, and pharmacological iron restriction improves, MDS pathophysiology in a preclinical study

AbstractAlthough iron overload is a common feature in myelodysplastic syndromes (MDSs), it remains unclear how iron excess is detrimental for disease pathophysiology. Taking advantage of complementary approaches, we analyzed the impact of iron overload and restriction achieved through genetic activation of ferroportin (FPN) via the C326S mutation (FPNC326S) and pharmacologic inhibition (vamifeport) of the iron exporter FPN, respectively, in a MDS mouse model. Although FPNC326S-induced iron overload did not significantly improve the late stages of erythroid maturation, vamifeport-mediated iron restriction ameliorated anemia and red blood cell maturation in MDS mice, through the reduction of oxidative stress and apoptosis in erythroid progenitors. Iron overload aggravated, and restriction alleviated, reactive oxygen species formation, DNA damage, and cell death in hematopoietic stem and progenitor cells, resulting in altered cell survival and quality. Finally, myeloid bias, indicated by expanded bone marrow myeloid progenitors and circulating immature myeloid blasts, was exacerbated by iron excess and attenuated by iron restriction. Overall, vamifeport treatment resulted in improved anemia and significant survival increment in MDS mice. Interestingly, the combined therapy with vamifeport and the erythroid maturation agent luspatercept has superior effect in improving anemia and myeloid bias as compared with single treatments and offers additive beneficial effects in MDS. Our results prove, to our knowledge, for the first time in a preclinical model, that iron plays a pathologic role in transfusion-independent MDS. This is likely aggravated by transfusional iron overload, as suggested by observations in the FPNC326SMDS model. Ultimately, the beneficial effects of pharmacologic FPN inhibition uncovers the therapeutic potential of early prevention of iron toxicity in transfusion-independent MDS.

M6A-modified exosome-derived circHIF1α binding to KH domain of IGF2BP3 mediates DNA damage and arrests G1/S transition phase to resists bacterial infection in bacteremia

BackgroundAnimal and human health are seriously threatened by bacterial infections, which can lead to bacteremia and extremely high rates of morbidity and mortality. Recently, there have been reports indicating the involvement of exosomal circular RNAs (circRNAs) in a range of human disorders and tumor types. However, the role of exosomal circRNAs in bacterial infection remains elusive.MethodsWe extracted and identified exosomes from the culture medium of PIEC cells infected with or without Glaesserella parasuis. RNA sequencing analysis was performed on the exosomes to screen and identify circRNAs (circHIF1α) associated with Glaesserella parasuis infection. PIEC cells were infected with Staphylococcus aureus or Streptococcus suis 2 to further determine whether exosome-derived circHIF1α was the crucial circHIF1α associated with bacterial infections. The transmission process of exosomes and their circHIF1α between cells was clarified via exosome tracing and co-culture assay. Moreover, the mechanism of circHIF1α being packaged into exosomes was explored, and the effects of exosomes and their circHIF1α on cell proliferation, DNA damage and cell cycle were analyzed. In addition, the binding mode and site of interacting proteins with circHIF1α were further determined. In vivo and in vitro, the role of exosomes and their circHIF1α in host resistance to bacterial infection was confirmed.ResultsWe first discovered a new circHIF1α that was very stable and detectable, encapsulated into exosomes by hnRNPA2B1, and whose expression in exosomes of bacterially infected PIEC cells significantly decreased. Additionally, exosomal circHIF1α reduced bacterial infection both in vitro and in vivo and suppressed the growth of reception cells. Mechanistically, the circHIF1α interacted with the KH domain of IGF2BP3 in an m6A-modified manner, which mediated DNA damage to arrest the cells at the G1/S phase through the interaction between the regulator of Chromosome Condensation 2 (RCC2) and γ-H2AX protein. Exosomal circHIF1α is a unique therapeutic target for bacterial infection since this work highlights its critical function in fighting bacterial infection.

Neurotoxicity, Cytotoxicity, and Genotoxicity of Phyto-radio Synthesized Selenium Nanoparticles in Culex pipiens Complex.

Effective mosquito management strategies are crucial to minimize the number of mosquito-borne diseases. Selenium nanoparticles (SeNPs) are promising in mosquito control because they are effective and eco-friendly rather than synthetic insecticides. The current study was conducted to evaluate the impact of SeNPs on the detoxification enzymes, acetylcholine esterase (AChE), glutathione S-transferase (GST), and α-carboxyl esterase (α-CarE), in larval instars of Culex pipiens complex at the LC50 concentration. In 3rd instar larvae treated with microwave-assisted selenium nanoparticles (SeNPs-MW) and gamma-assisted selenium nanoparticles (SeNPs-G), it was found that AChE activity was significantly inhibited. On the other hand, significant increases in GST and α-CarE activities were observed. Additionally, genotoxic and ultrastructure studies of midgut epithelial cells in 3rd instar larvae revealed DNA damage and cell lysis, including destruction of the cell membrane, microvilli, and nuclei. These findings suggest that SeNPs have an adverse effect on AChE gene expression, resulting in its downregulation. This downregulation can be attributed to the formation of reactive oxygen species induced by SeNPs that can modulate the host defense mechanism leading to apoptosis and subsequent larval mortality. The present study was the first to use phyto-microwave-assisted and gamma-assisted synthesis of SeNPs which provides an eco-friendly and cost-effective solution to reduce the risk of chemical insecticides. Furthermore, an integrated pest management program (IPM) using nanocides can be successfully developed for mosquito control.

Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks.

DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs)and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin(SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids andefficient DNA repair.

PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling.

RNF168 orchestrates a ubiquitin-dependent DNA damage response to regulate the recruitment of repair factors, such as 53BP1 to DNA double-strand breaks (DSBs). In addition to its canonical functions in DSB signaling, RNF168 may facilitate DNA replication fork progression. However, the precise role of RNF168 in DNA replication remains unclear. Here, we demonstrate that RNF168 is recruited to DNA replication factories in a manner that is independent of the canonical DSB response pathway regulated by Ataxia-Telangiectasia Mutated (ATM) and RNF8. We identify a degenerate Proliferating Cell Nuclear Antigen (PCNA)-interacting peptide (DPIP) motif in the C-terminus of RNF168, which together with its Motif Interacting with Ubiquitin (MIU) domain mediates binding to mono-ubiquitylatedPCNA at replication factories. An RNF168 mutant harboring inactivating substitutions in its DPIP box and MIU1 domain (termed RNF168 ΔDPIP/ΔMIU1) is not recruited to sites of DNA synthesis and fails to support ongoing DNA replication. Notably, the PCNA interaction-deficient RNF168 ΔDPIP/ΔMIU1 mutant fully rescues the ability of RNF168-/- cells to form 53BP1 foci in response to DNA DSBs. Therefore, RNF168 functions in DNA replication and DSB signaling are fully separable. Our results define a new mechanism by which RNF168 promotes DNA replication independently of its canonical functions in DSB signaling.

Parishin Alleviates Pulmonary Fibrosis by Reducing CD38 Levels in Naturally Aging Mice.

Parishin, a natural compound, has demonstrated significant potential in mitigating age-related phenotypes and improving outcomes in age-associated diseases. Given that aging is a major risk factor for numerous chronic conditions, including pulmonary fibrosis, we investigated parishin's effects on cellular senescence and lung health. In our study, we treated mouse lung epithelial cells with parishin and observed a reduction in cellular senescence markers alongside an upregulation of sirtuin 1 (SIRT1). Building on these in vitro findings, we administered parishin to naturally aged mice. The treatment resulted in decreased pulmonary fibrosis and reduced DNA damage in lung tissue. Notably, we found that parishin treatment led to a reduction in Cluster of differentiation 38 (CD38) levels, concomitant with an increase in SIRT1 expression. These findings indicate that parishin may enhance lung function in aged mice, suggesting its potential as a therapeutic agent for treating age-related pulmonary disorders.

Branching NOTCH1 to DNA damage in T-cell acute lymphoblastic leukemia.

Not available.

Mechanisms of Staphylococcus aureus survival of trimethoprim-sulfamethoxazole-induced thymineless death.

Trimethoprim-sulfamethoxazole (SXT) is commonly used to treat diverse Staphylococcus aureus infections, including those associated with cystic fibrosis (CF) pulmonary disease. Studies with Escherichia coli found that SXT impairs tetrahydrofolate production, leading to DNA damage, stress response induction, and accumulation of reactive oxygen species (ROS) in a process known as thymineless death (TLD). TLD survival can occur through the uptake of exogenous thymidine, countering the effects of SXT; however, a growing body of research has implicated central metabolism as another potentially important determinant of bacterial survival of SXT and other antibiotics. Here, we conducted studies to better understand the mechanisms of TLD survival in S. aureus. We found that thymidine abundances in CF sputum were insufficient to prevent TLD of S. aureus, highlighting the importance of alternative survival mechanisms in vivo. In S. aureus cultured in vitro with SXT and low thymidine, we frequently identified adaptive mutations in genes encoding carbohydrate, nucleotide, and amino acid metabolism, supporting reduced metabolism as a common survival mechanism. Although intracellular ROS levels rose with SXT treatment in vitro, survival was not improved in the presence of ROS scavengers, unlike in E. coli. SXT challenge induced the SOS response, which was alleviated by added thymidine. Finally, an inactivating mutation in the phosphotransferase gene ptsI conferred both limitation in cellular ATP and improved survival against TLD. Collectively, these results suggest that alterations in core metabolic functions, particularly those that reduce ATP levels, predominantly confer S. aureus survival and persistence during SXT treatment, potentially identifying novel targets for co-treatment.IMPORTANCEStaphylococcus aureus is a ubiquitous organism and one of the leading causes of human infections, many of which are difficult to treat due to persistence, antibiotic resistance, or antibiotic tolerance. As our arsenal of effective antibiotics dwindles, the need for improved treatments becomes increasingly urgent, necessitating a better understanding of the precise mechanisms by which pathogens evade our most critical antimicrobial agents. Here, we report a systematic characterization of the mechanisms of S. aureus survival to treatment with the first-line antistaphylococcal antibiotic trimethoprim-sulfamethoxazole, identifying pathways and candidate targets for enhancing the efficacy of available antimicrobial agents.