DNA Repair

Artificially Expanded Genetic Information Systems (AEGIS) increase the information content of nucleic acids by including new nucleobase pairings that are orthogonal to those of canonical Watson-Crick nucleobases. DNA ligases do not form direct interactions with the nucleobases during catalytic turnover, suggesting that these enzymes should efficiently and faithfully join double-stranded AEGIS substrates. Here we report the systematic investigation into the validity of this hypothesis for structurally-diverse DNA ligases employing substrates built from the eight nucleotide hachimoji genetic alphabet, where orthogonality is achieved by rearranging the hydrogen bonding patterns seen in canonical Watson-Crick pairs. We find that single, or multiple, non-canonical bases are well tolerated at the 5 prime-end of the nick. However, tracts of consecutive non-canonical bases at the 3 prime-end of the break significantly decrease ligation efficiency or abolish it altogether. Possible reasons for this apparent bias against non-canonical nucleobases could include incompatibility in electrostatic interactions between the ligase active site and the non-canonical substrates or altered conformational preferences and/or dynamics in key catalytic intermediates. We also observe single hachimoji mismatches are ligated more frequently than mis paired canonical bases, potentially due to promiscuous pairing of tautomeric forms of the non-canonical bases.

Api5 is elevated in a number of cancers and is associated with many hallmarks of cancer, including resistance to apoptosis, immune escape, stemness, chemotherapy resistance, high proliferation, and cell-cycle dysregulation. In this study, we identified the DNA and chromatin-binding activities of Api5 in tumorigenic cells, as well as its association with genomic instability and chemotherapy resistance. Knockdown of Api5 resulted in reduced nuclear volume, DNA content, and chromosome number, and increased sensitivity to DNA damage. The survival of Api5-knockdown cells decreased following UV and cisplatin treatments due to the accumulation of damaged DNA and inefficient nucleotide excision repair. Interestingly, Api5 knockdown cells also exhibited low pChk1 levels following UV damage. Further, we confirmed the chemotherapy resistance phenotype in cancers with elevated Api5 levels, demonstrating that xenograft tumours with Api5 knockdown responded better to cisplatin, with significant tumour regression. SummaryApoptosis inhibitor 5 (Api5) contributes to chemotherapy resistance by conferring a survival advantage and promoting efficient DNA repair following genotoxic stress through regulation of Chk1 activation.

Bacterial survival after ultraviolet (UV) exposure is shaped not only by the extent of DNA damage but also by the physiological state-dependent capacity for DNA repair. Here, we examined the mechanisms underlying growth phase-dependent UV resistance in Escherichia coli K-12 exposed to 262 nm UV irradiation. Stationary-phase cells required higher UV fluence for log inactivation than exponential-phase cells, whereas the levels of UV-induced DNA damage, assessed by cyclobutane pyrimidine dimer staining and real-time PCR, did not differ markedly between the two growth phases. Deletion of nucleotide excision repair (NER) genes, including uvrA, uvrB, uvrC, and uvrD, markedly reduced survival after UV irradiation, indicating that NER is essential for the high UV resistance of stationary-phase cells. Quantitative real-time reverse transcription PCR showed stronger UV-induced expression of several DNA repair and UV resistance genes, including uvrA, uvrB, cho, umuC, and umuD, in stationary-phase cells than in exponential-phase cells. Furthermore, deletion of the DNA cytosine methyltransferase gene dcm increased UV resistance and enhanced the expression of uvrB, cho, umuC, umuD, and sulA in stationary-phase cells. These findings suggest that DNA cytosine methylation modulates UV resistance in E. coli, at least in part by influencing NER- and SOS-associated gene expression.

The histone variant H2A.J is implicated in radiation-induced senescence by promoting the transcription of inflammatory genes. While H2A.J expression increases in renal tubular epithelial cells (TECs) following ionizing radiation (IR), its functional role remains poorly understood. To investigate this, constitutive H2A.J knock-out (KO) mice and wild-type (WT) controls were subjected to CT-guided IR (fractionated whole-body or localized kidney IR). Kidneys were analyzed at acute, intermediate, and chronic stages using immunofluorescence, histochemistry, automated image analysis, and electron microscopy. In WT TECs, IR induced rapid chromatin incorporation and C-terminal serine phosphorylation of H2A.J. Conversely, KO kidneys exhibited significantly more severe histopathological damage, including tubular dilation, flattened epithelium, associated with increased apoptosis, and premature senescence, characterized by persistent DNA damage with lamin B1 loss. Notably, KO TECs displayed disrupted mitochondrial networks and reduced brush borders even at baseline, which were further exacerbated by IR. Unlike WT controls, KO kidneys developed progressive tubular atrophy and incipient fibrosis, indicating a failure in regenerative capacity. Our findings suggest that H2A.J loss impairs tubular regeneration due to defective mitochondrial activation, resulting in insufficient energy supply for coordinated repair. Collectively, these results identify H2A.J as a critical stress-adaptive histone variant essential for the epigenetic regulation of tissue repair following radiation-induced damage. One Sentence SummaryIn irradiated kidney, the loss of histone variant H2A.J impairs the chromatin-mediated adaptation of mitochondrial function in tubular epithelial cells, thereby exacerbating cellular stress - characterized by increased induction of apoptosis and senescence - and ultimately leading to tubular atrophy. Translational RelevanceAcute and chronic kidney injury are frequent complications of genotoxic cancer therapies. Chemo- and radiotherapy induce DNA lesions that trigger cell death and senescence, often leading to irreversible renal damage. However, renal regeneration can occur through the dedifferentiation, proliferation, and redifferentiation of surviving tubular epithelial cells (TECs). This repair process is governed by epigenetic mechanisms that regulate the DNA damage response (DDR) and adapt gene expression programs. Following ionizing radiation (IR), epigenetic remodeling involves the incorporation of histone variants that modulate chromatin accessibility for stress-responsive transcription factors. We identify the histone variant H2A.J as a constitutive component of renal TECs, significantly upregulated after exposure to ionizing radiation (IR). Using H2A.J knock-out (KO) mice, we demonstrate that its absence disrupts acute damage responses and prevents coordinated repair, severely impairing regeneration. Mechanistically, H2A.J deficiency compromises mitochondrial function under postirradiation metabolic stress, driving the transition from acute injury to chronic kidney disease via persistent inflammation and maladaptive tubulointerstitial repair. Targeting these epigenetic drivers offers a promising strategy for regenerating damaged kidney tissue in oncology.

Radiation exposure induces systemic and cellular damage, contributing to acute radiation syndrome and long-term effects such as premature aging and carcinogenesis. At the cellular level, radiation triggers apoptosis, mutation, and transformation through oxidative damage and activation of pathways including ER stress-mediated autophagy. Autophagy plays a context-dependent dual role in stressed cells, but its contribution to intestinal recovery after acute radiation remains unclear. Here, we evaluated combinatorial radiomodification using gamma radiation (8 Gy) and autophagy modulators in whole-body irradiated C57BL/6 mice (8-10 weeks old, n = 10). Mice were treated with autophagy inducers or inhibitors and euthanized at 3-, 8-, and 30-day post-irradiation. The jejunal-ileal region was analyzed via antioxidant assays, immunoblotting, H&E staining, and immunohistochemistry. Radiation significantly altered oxidative stress and autophagy markers, including increased LC3-II and decreased SQSTM1/p62. Autophagy induction enhanced intestinal proliferation (as measured by Ki-67), whereas inhibition impaired regeneration. Rapamycin pretreatment improved survival and reduced markers of intestinal injury following 8 Gy total body irradiation (TBI), whereas chloroquine exacerbated several injury-associated parameters. Overall, our findings suggest that targeted modulation of autophagy is a promising strategy for alleviating radiation-induced gastrointestinal injury and provide mechanistic insights relevant to therapeutic development.

Abstract Introduction: IL-15 is one of the most promising candidate cytokines in cancer immunotherapy due to its ability to promote the activity of different cytotoxic innate immune cell subsets such as NK, ILC1 and gammadelta T cells. During biosynthesis, IL-15 associates with IL-15alpha and is transported to the cell surface where IL-15Ralpha trans-presents IL-15 to target neighboring cells expressing the beta chain (IL-2Rbeta) and the common gamma chain. Our group previously showed that in autoimmune type 1 diabetes and early innate immune responses to infections trans-presentation by IL-15Ralpha is dispensable. Here we addressed the relative roles of IL-15 and trans-presented IL-15 in the control of established tumors and spontaneous tumor development. Methodology: Growth kinetics of tumor cell lines were monitored in WT, Il15-/- and Il15ra-/- mice. Spontaneous fibrosarcoma was induced with Methylcholanthrene (MCA) in WT, Il15-/- and Il15ra-/- mice. Cell lines were established from MCA-induced tumors to characterize their immunogenicity. Results: Growth of established tumor cell lines were comparable in the three genotypes. MCA-induced tumor incidence was reduced in Il15ra-/- mice when compared to WT and Il15-/- mice. In vitro, MCA tumor-derived cell lines expressed MHC-I and PD-L1 and had comparable proliferation rates. In vivo, MCA tumor-derived cell lines established from the 3 genotypes showed comparative growth in WT mice suggesting that IL-15 does not impact immunoediting. Nonetheless, NLRC5 expressing B16-F10 tumors were contained in WT and Il15ra-/- mice but not in Il15-/- mice. Conclusions: Taken together, these results show that in the absence of trans-presentation by IL-15Ralpha, IL-15 can better control spontaneous tumor development and that IL-15 signaling plays a minor role in immunosurveillance in this model. IL-15 signaling, independent of IL-15Ralpha has a significant role in the control of solid tumors.

Symmetry elements in nucleic acids are most strongly correlated with sites of biological function; however, their relevance to non-canonical structures remains underexplored. In this study, we demonstrate the presence and significance of trinucleotide symmetry elements within G-quadruplex (G4) motifs. Our central hypothesis is that the intra-strand mirror symmetry of trinucleotides has been evolutionarily selected to facilitate G4 formation builds on the established sequence-structure association of G-quadruplexes and the natural symmetry law governing nucleotide insertion during genome evolution. Using a conserved G4 motif in the first exon of the MTOR gene as a model, we showed remarkable trinucleotide symmetry preservation across primates and broader mammals, with functional G4 regions displaying locally elevated symmetry relative to the codon-biased exonic background. Analysis of experimentally validated oncogenic G4s, including c-MYC, BCL2, VEGF, and KRAS, revealed that mirror and reverse complement symmetries converge around biologically important G4s. To quantify this feature, we formulated two complementary descriptors: the mirror symmetry index (MSI) and its non-palindromic variant (nMSI). Across 14 oncogene-promoter wild-type G4s, the majority scored MSI [≥] 0.80 (mean 0.884), with only the loop-rich ATG7, BCR, and MDM2 motifs falling below this value, and the KRAS promoter G4 reached individual significance against its mononucleotide-preserving null distribution (p = 0.042). Most decisively, each wild-type G4 scored higher on MSI than its experimentally confirmed G4-abolished mutant in 12 of 14 paired comparisons (sign test, p = 0.0065; mean {Delta}MSI = +0.089, mean {Delta}nMSI = +0.192); the two reversals (BCL2 and HIF-1) are attributable to scrambled mutant controls that introduce more balanced trinucleotide compositions rather than to failure of the index. The directional trend was reproduced across three independently published datasets, with nMSI [≥] 0.50 separating G4-forming from non-G4 sequences at 77.8% sensitivity and 100% specificity, although the collective per-sequence signal from mononucleotide-preserving shuffles remained a non-significant trend (Stouffer combined Z = 1.197, p = 0.116). This first report of trinucleotide symmetry in G4 motifs posits that coordinated nucleotide insertion and quadruplet maintenance act as an evolutionary forcing mechanism that pre-organizes single strands for G4 folding.

Thermostable DNA polymerases are indispensable tools in molecular biology, yet enzymes from the most extreme hyperthermophiles remain largely uncharacterized. Here, we report the biochemical and structural characterization of a family B DNA polymerase from Pyrolobus fumarii A1 (Pyrfu pol), one of the most thermoresistant archaea described to date. The enzyme was efficiently overproduced in E. coli Rosetta 2(DE3)[pLysS] and purified to homogeneity using a two-step protocol that combined heat treatment with immobilized metal affinity chromatography (IMAC). Bioinformatic analysis confirmed the canonical family B architecture, while AlphaFold-based structural modeling and comparative analysis with mesophilic RB69 DNA polymerase revealed a well-conserved structural core alongside thermoadaptive features. Radiolabel incorporation assays demonstrated enzymatic activity over a broad ionic strength range and an absolute requirement for Mg ions. PCR-based optimization confirmed these findings and revealed broad pH tolerance (6.5-11.0). Notably, Tris inhibited radiolabel-based assays (pH 7.0) yet proved essential for efficient PCR amplification (pH 8.5), suggesting a context-dependent role of buffer composition in polymerase activity. Processivity assays confirmed amplification of DNA fragments up to approximately 8,000 bp. Replication fidelity, assessed by the lacZ-based assay, showed a 2.9-fold improvement over Taq polymerase. Urea-nanoDSF yielded an exceptional melting temperature of 105.9 {+/-} 0.08 {degrees}C. Pyrfu pol also demonstrated tolerance to common PCR inhibitors, highlighting its potential utility in molecular biology applications.

Bacterial deubiquitinases (DUBs) are important virulence effectors that manipulate host ubiquitin signaling during infection. ElaD, a CE-clan DUB expressed by enterohemorrhagic Escherichia coli, preferentially cleaves K63-linked ubiquitin chains, yet its effects on conserved cellular stress responses remain poorly understood. We demonstrate that ElaD exhibits redox-dependent DUB activity in vitro. In addition, we identified the molecular basis underlying the selective recognition of substrate proteins, ubiquitin and NEDD8 by ElaD. Structural and mutational analyses reveal that, beyond the conserved catalytic site, ElaD engages ubiquitin through a combination of electrostatic and hydrophobic interactions. Using Saccharomyces cerevisiae as a heterologous model system, we show that wild-type ElaD rescues the proteotoxic stress phenotype of ubp2{Delta} yeast cells, whereas specific ElaD mutants fail to confer a similar response. Furthermore, expression of ElaD suppresses oxidative stress-induced accumulation of K63-linked polyubiquitin and may perturb stress-associated translational regulation linked to K63 ubiquitin signaling. Consequently, cells expressing ElaD exhibit altered stress adaptation and diminished fitness during prolonged oxidative stress. Collectively, these findings indicate that ElaD perturbs ubiquitin-mediated stress signaling by counteracting K63-linked ubiquitination events that support adaptive cellular responses. Our study highlights how a bacterial DUB can reprogram conserved ubiquitin-dependent pathways and exploit host ubiquitin signaling networks to modulate cellular stress responses and protein homeostasis. These findings further suggest potential host targets of bacterial DUBs during infection.

Yes-associated protein (YAP), a transcriptional coactivator, plays key roles in cell growth, proliferation and apoptosis, and its levels are frequently dysregulated in cancers. YAP levels in the nucleus are highly sensitive to nuclear mechanical cues, and such cues are also parallelly emerging to be a key modulator of DNA Damage Responses (DDR). However, whether DNA-damage can induce mechanical changes that regulate downstream events such as YAP localization and that in turn feeds back onto DDR activation, remains unknown. In this study, we report that YAP translocates in a nuclear mechanics-dependent manner upon induction of Double Strand Breaks (DSBs). This translocation is not a mere epiphenomenon, and we find that: first, global nuclear eviction of YAP enhances DDR signaling; second, local enrichment of YAP at DNA damage sites promotes recruitment of DNA repair proteins previously identified as potential interactors of YAP or its partner TEAD1. Together, these findings indicate that YAP is not only a transcriptional coactivator, but also plays an under-appreciated role in regulating DDR.

Cell-cycle checkpoints couple cell division to environmental and intracellular stress. Here, we show that the human fungal pathogen Cryptococcus neoformans possesses two divergent Wee1-family kinases, CnSwe1 and CnSwe102, that retain conserved CDK-inhibitory activity but function differently from their counterparts in the canonical Saccharomyces cerevisiae morphogenesis checkpoint. The swe1{Delta} and swe102{Delta} mutants displayed distinct stress-response defects, and genetic analyses suggested a dosage-sensitive genetic interaction between SWE1 and SWE102. Although both proteins promoted Cdc28 tyrosine phosphorylation and elongated-cell morphology when expressed in S. cerevisiae, neither localized to the mother-bud neck in C. neoformans. Altered SWE1 dosage in the absence of SWE102 increased sensitivity to replicative and DNA-damaging stresses and perturbed cell-cycle progression under genotoxic conditions. Importantly, loss of SWE1 nearly abolished virulence in a murine infection model, and SWE102 also contributed to pathogenicity. Together, these findings indicate that the conserved Wee1-CDK module acts in C. neoformans as a dosage-sensitive checkpoint that promotes stress adaptation and fungal virulence.

Cyclin-dependent kinases (CDKs) control eukaryotic cell-cycle progression by phosphorylating specific substrates with substrate recognition often involving cyclin-specific docking interactions. However, in minimal cell cycle control systems driven by a single cyclin-CDK complex, how docking interactions contribute to the differential timing of substrate phosphorylation remains unclear. Here, we used AlphaFold-Multimer to systematically predict interactions between the fission yeast mitotic cyclin-CDK fusion Cdc13-L-Cdc2 and its known in vivo CDK substrates. We found that many substrates are predicted to interact with the cyclin hydrophobic patch, and have identified a previously uncharacterised docking motif, [FVIPWGLAM](x)xER[LMV] (ERL motif), with features consistent with an atypical RxL motif. We show that ERL motifs can functionally substitute for canonical RxL motifs to promote phosphorylation of a model CDK substrate by Cdc13-Cdc2, while the S-phase cyclin-CDK Cig2-Cdc2 was found to preferentially phosphorylate substrates containing canonical RxL motifs. Finally, we investigated whether Cdc13-L-Cdc2 is predicted to preferentially bind DNA replication substrates over mitotic substrates but found no evidence of differential binding. These results reveal diversity in cyclin-CDK substrate recognition beyond established docking motifs.

DNA double-strand breaks (DSBs) are threats to genome integrity, and to mitigate this risk cells activate the Ataxia Telangiectasia and Rad3-related (ATR) kinase, which halts cell cycle progression to allow time for repair. While ATR signalling during replication stress is well understood, how ATR is activated at DSBs remain unclear. Topoisomerase 2-Binding Protein 1 (TOPBP1) is a key activator of ATR, and activation is mediated by phosphorylation of TOPBP1 at Serine 1131 (S1131). Previous work showed that the Ataxia Telangiectasia Mutated (ATM) kinase phosphorylates TOPBP1 at S1131. ATM is primarily linked to the homologous recombination (HR)-based repair of DSBs, however the majority of cellular DSBs are repaired via the Non-Homologous End-Joining (NHEJ) repair pathway, raising the question of how (or if) ATR is activated in an ATM-independent manner. Here, using Xenopus egg extracts, we demonstrate that DNA-PKcs controls a pathway acting in parallel to ATM that promotes ATR signalling at DSBs. We show that, like ATM, DNA-PKcs phosphorylates TOPBP1 at S1131. DNA-PKcs is best known for orchestrating NHEJ, however we find that its roles in NHEJ and ATR signalling are separable. Our findings reveal an alternative pathway for ATR activation in which DNA-PKcs directly couples DSB recognition to ATR signalling. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/733426v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@4eb847org.highwire.dtl.DTLVardef@244193org.highwire.dtl.DTLVardef@4d4f4forg.highwire.dtl.DTLVardef@191de27_HPS_FORMAT_FIGEXP M_FIG C_FIG

Premature termination of transcription (PTT), also known as attenuation, is a conserved gene regulatory mechanism that operates across all domains of life and in viruses. Attenuation enables rapid cellular responses to environmental and metabolic changes and fine-tunes expression of biosynthetic genes. In Saccharomyces cerevisiae, attenuation of RNA Polymerase II (Pol II) transcription was first linked to the Nrd1-Nab3-Sen1 (NNS) termination pathway for non-coding RNAs, and the mRNA 3-end processing factor Hrp1 has been implicated more recently. Substitutions in Hrp1 RNA Recognition Motifs (RRMs) cause attenuator readthrough and reduce RNA-binding affinity in vitro, but direct evidence for Hrp1 functioning at attenuators in vivo remains limited. Here, we characterized 5-end RNA terminator elements from several genes, including RAD3, SNG1, MNR2, and CPR8. Readthrough mutations clustered in AU-rich regions resembling polyadenylation site (pA) efficiency elements, consistent with Hrp1 binding targets. Amino acid substitutions of Hrp1 RRM residue F162 revealed a general requirement for aromaticity in RNA recognition that varied to some degree by gene context. To test Hrp1-RNA interactions independent of other yeast factors, we adapted a bacterial 3-hybrid (B3H) assay. Hrp1 interacted with RNA derived from the GAL7 3-end pA site and 5-end terminator regions of RAD3, MNR2, and CPR8. Mutations in AU-rich RNA regions that disrupted Pol II attenuation in yeast generally impaired B3H interactions. However, some Hrp1 mutants (M191T, I270T, D271G, M275V, T280I) retained binding to CPR8 terminator RNA, suggesting their defects require additional yeast components. These results demonstrate that Hrp1 is sufficient to bind multiple UA-rich attenuator RNAs in vivo, expanding Hrp1 function to include early transcription events.

Structural variants (SVs) in cancer are associated with defects in DNA repair and replication stress, but the mechanisms generating common SV types remain unresolved. We propose that large (>100 kb) tandem duplications originate through a novel sister-fork breakage-fusion mechanism. To capture replication-related context beyond breakpoints, we developed an algorithm to characterize replication timing, origin density, and fork direction across SV-spanned regions, features that refine and differentiate previously defined SV signatures. Large tandem duplications frequently overlap replication origins from which forks proceed bidirectionally; combined with independent evidence from APOBEC strand asymmetry, this pattern is compatible uniquely with the proposed mechanism. Although tandem duplications in CCNE1-amplified and CDK12-mutant cancers also concentrate around origins and highly transcribed genes, they display distinct contexts: CDK12-mutant SVs arise near later-firing origins, whereas those in CCNE1--amplified tumors often coincide with genes in specific strand configurations, suggesting different causes of fork stalling. Incorporating replication features into signature analysis enabled the discovery of new SV signatures, which we used to build SVIG, a multi-class classifier of SV phenotypes. SV signatures attributed to replication stress may help guide therapies targeting this vulnerability.

Protein homeostasis - i.e., proteostasis - is the biological process by which the qualitative and quantitative balance of the proteome is conducted, either by preserving functionally relevant proteins or by degrading unnecessary ones. Stress conditions can modulate cellular proteostasis in order to promote cytoprotection and preserve the viability of living organisms. Among the cellular pathways already described that can play an important role in preserving biological functions by modulating proteostasis are the heat shock response and the ribosome quality control pathways. In this work, we show that the Rqc1p protein is necessary for the thermoadaptation of S. cerevisiae to heat shock, as RQC1-deficient yeast is sensitive to elevated temperatures. In silico approaches - such as multiple sequence alignment, structural analysis, and molecular dynamics simulations - confirmed earlier predictions that Rqc1p shares characteristics with the bHLH family of proteins. We also verified, through computational prediction of sub-cellular localization, that S. cerevisiae Rqc1p contains nuclear localization signals, suggesting that this protein can potentially be translocated toward the nucleus, thereby broadening its current range of recognized biological functions in this organism. Also, analysis of yeast transcriptomes subjected to heat shock showed that Rqc1p mRNA levels do not fluctuate in response to heat shock, suggesting that cellular concentrations of Rqc1p are already at optimal levels to elicit a rapid and effective response during thermal stress in S. cerevisiae.

[FeFe]-hydrogenases are very active biocatalysts for H2 conversion. However, their active site is vulnerable to irreversible degradation initiated by O2 binding at the catalytic iron ion (Fed) of the active center. CbA5H, the [FeFe]-hydrogenases from Clostridium beijerinckii exhibits stability towards oxygen (O2) due to its ability to reversibly enter an inactive state termed Hinact upon contact with O2. We previously proposed that the close distance of approximately 3.1 [A] between the thiol of a nearby cysteine (C367) and the Fed, based on a 2.9 [A] crystal structure of CbA5H in the Hinact state, enables their binding to each other. This binding therefore was suggested to shield the Fed from O2 damage. However, there is currently a lack of evidence to support this hypothesis. Furthermore, density functional theory (DFT) calculations based on a homologous model favored hydroxide as the binding ligand of the Fed over the thiol of C367. In this study, we present the crystal structure of CbA5H in the Hinact state at an improved resolution of 2.15 [A]. The structure reveals a direct binding between the thiol of C367 and the Fed with a distance of approximated 2.77 [A] which is well supported by our DFT calculations based on the new crystallographic data. It is noteworthy that the 2.77 [A] bond distance is strikingly long when compared with other iron-sulfur bonds. This finding may provide a crucial foundation for understanding the rapid reversibility of the Hinact state.

G-quadruplexes (G4s) are non-canonical DNA secondary structures that can impede DNA replication and transcription and provoke genome instability, and DNA helicases of the PIF1 and RecQ families have long been regarded as the principal enzymes that resolve them. To directly test the relative contributions of these families, we measured the growth of Saccharomyces cerevisiae helicase mutants in the presence of the G4-stabilizing ligand pyridostatin (PDS). Unexpectedly, no single PIF1- or RecQ-family mutant was sensitized to PDS relative to wild type. Sensitivity emerged only in double mutants, and it did so for combinations both within a single family and across the two families. This pattern indicates that G4 tolerance is buffered by the combined, partially interchangeable, activity of multiple helicases rather than by any one family. To ask whether this redundancy extends beyond the canonical players, we tested two additional helicases whose human orthologs are implicated in G4 metabolism: Chl1 (DDX11/ChlR1) and Srs2 (RTEL1). Loss of Chl1 alone did not sensitize cells, and chl1{Delta} combined with PIF1- or RecQ-family mutations recapitulated the redundancy pattern - with one informative exception: chl1{Delta} hrq1{Delta} remained PDS-tolerant, placing Chl1 and Hrq1 in a shared genetic route. In contrast, srs2{Delta} was the sole single mutant sensitized to PDS, defining a non-redundant requirement that no other helicase compensates. We integrate these results into a two-layer model in which a redundant helicase pool resolves G4-associated genomic stress, while a non-redundant Srs2 function manages its recombinogenic consequences. Our findings reframe G4 maintenance from a family-specific activity into a distributed, buffered network. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/737069v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@18db695org.highwire.dtl.DTLVardef@ce0062org.highwire.dtl.DTLVardef@7b2a8corg.highwire.dtl.DTLVardef@267073_HPS_FORMAT_FIGEXP M_FIG C_FIG ARTICLE SUMMARYDNA helicases, enzymes that unwind DNA, are thought to dismantle G-quadruplexes (G4s), four-stranded DNA structures that can block DNA metabolism and destabilize genomes. In Saccharomyces cerevisiae, we used the chemical pyridostatin to stabilize G4s and measured the growth of helicase mutants. Losing any single helicase had no effect, but losing two together - even from different helicase families - impaired growth. The protein Chl1 works with the helicase Hrq1 in one shared pathway, while Srs2 is uniquely required on its own. G4 tolerance therefore depends on a redundant network of helicases. These findings interest researchers studying genome stability and related human cancer-predisposition disorders.

Some key inflammatory genes controlled by the RELA transcription factor are thought to be highly expressed in fibroblasts induced into senescence by ionizing radiation (IR) as part of the Senescent-Associated Secretory Phenotype (SASP). However, this view is based largely on studies of a limited number of fibroblast cell lines derived from fetal lung or neonatal foreskin. Here, we show that more than half of the primary adult fibroblast strains examined exhibit only weak induction of RELA-dependent inflammatory genes following IR-induced senescence. We define these fibroblasts as "low-responding" to distinguish them from fibroblasts that express high levels of inflammatory gene expression in response to IR. RNA-seq analysis indicated particularly weak IL1A and IL1B expression in low-responding fibroblasts. IL1-alpha and IL1-beta participate in a positive amplification loop for inflammatory gene expression in senescence. Addition of recombinant IL1-alpha or IL1-beta to these fibroblasts sufficed to induce high expression of inflammatory genes. Low-responding fibroblasts thus exhibit cell-autonomous defects in IL1A and IL1B gene activation in response to IR that explains their overall low expression of RELA-targeted inflammatory genes. This defect was correlated with reduced chromatin accessibility and H3-K27-acetylation at 2 putative enhancers in the intergenic region separating IL1A and IL1B, and deletion of either of these enhancers inhibited inflammatory gene expression in IR-induced senescence. Fibroblasts express distinct transcriptomes and we found that differential expression of the FOXF1 transcription factor gene in high-responding WI38 fetal lung fibroblasts contributes to inflammatory gene expression after IR. Our observations indicate that fibroblasts can be distinguished by their ability to manifest cell-autonomous induction of inflammatory genes under conditions of IR-induced senescence.

Anthracyclines are highly effective chemotherapeutic agents that cause DNA and chromatin damage. One member of the anthracyclines, aclarubicin, has recently gained therapeutic interest due to its ability to kill cancer cells through chromatin-based mechanisms, thus avoiding the off-target effects associated with DNA damage. Despite this, the molecular mechanism of action leading to aclarubicin-induced chromatin damage remains elusive. Here we performed Cleavage Under Targets and Tagmentation (CUT&Tag) of RNA polymerase II (Pol II) and other transcriptional regulators in human cells during aclarubicin treatment. We found that aclarubicin strongly disrupts the replication-coupled histone genes, resulting in a nonproductive accumulation of Pol II-transcription machinery here beyond the levels at other genes. We attribute this sensitivity to the dense Pol II loading and rapid transcription of the histone genes, which intensify the chromatin-disrupting effects of aclarubicin at these loci. Together, our findings support the effectiveness of aclarubicin as an anticancer drug and point to the histone gene cluster as a promising target for therapeutic intervention.