DNA Repair

Whether the homologous parallel triplex (HP-triplex, formed by dsDNA and a homologous ssDNA) acts as an intermediate prior to DNA recombination remains controversy, owing to the intrinsic instability and lake of experimental data. Here, we report a HP-triplex that is stable at least at 35 oC under protein-free conditions. Two complementary circular ssDNAs were first hybridized at a 1:1 ratio to form an LR-chimera containing both Z-DNA and B-DNA. Then the circular homologous ssDNA (T-rich) was found to easily hybridize with this LR-chimera to form the HP-triplex (strand ratio of 2:1), involving both left- and right-handed triplex parts under the topological constraint. The third strand locates in the major groove and forms hydrogen bonds with both bases in each base pair of dsDNA without obvious strand exchange. Our data indicate that the third-strand ssDNA may first bind to the B-DNA region, followed by association with the Z-DNA region to assemble the full triplex structure. Structural simulations and analyses reveal that topological constraint imposed by the circular architecture raises the energy barrier for strand displacement, thereby stabilizing this triplex. In summary, the dynamic HP triplex can be stabilized via a Z B junction structure that mimics the in vivo one formed by negative supercoiling. These findings demonstrate that HP-triplex may act as an intermediate for DNA recombination, anti-gene strategy, and other interactions between dsDNA and homologous ssDNA or RNA.

Mammalian base excision repair (BER) is the major repair pathway for correcting small DNA base lesions. BER operates via two sub-pathways: short patch (or single nucleotide) and long patch BER (SP-BER and LP-BER, respectively). In LP-BER, DNA polymerase beta (Pol{beta}) performs strand-displacement synthesis generating a 5'-flap that is removed by flap endonuclease 1 (FEN1). To determine the precise DNA-intermediate that "switches on" FEN1 activity, we analyzed the FEN1 cleavage position in DNA-intermediates generated by Pol{beta}. For simultaneous detection of Pol{beta}-catalyzed primer elongation and FEN1's cleavage products, we utilized DNA structures with fluorescent labels on the upstream and downstream primers. Our data indicate that FEN1 operates efficiently on DNAs containing one or more equilibrium nucleotides. FEN1 binds to the 3'-terminal equilibrium nucleotide of the primer and probes it for a 3'-OH group. The cleavage point is located directly opposite this 3'-terminal equilibrium nucleotide of the primer. The subsequent elimination of the equilibrium area leads to restoration of the ligatable structure, thereby limiting further strand-displacement synthesis. Since the product sets generated by the two enzymes are mirror-symmetrical, we propose that pausing of Pol{beta} permits FEN1 to join the Pol{beta}-DNA complex. Our study demonstrates that the FEN1-Pol{beta} interplay occurs via the generation and elimination of equilibrium nucleotide areas rather than through a "gap-translation" mechanism. The length of the equilibrium area depends on dNTP concentration, suggesting that the size of the LP-BER repair patches may differ between S-phase and the remainder of the cell cycle.

Chlorella sorokiniana is an industrially important microalga, but its genetic manipulation remains limited by poor transformation efficiency. Although several barriers have been proposed, the role of intracellular nuclease activity has received little attention. Here, we compared the degradation of exogenous double-stranded DNA in whole-cell lysates from transformation-recalcitrant (Chlorella sorokiniana and Tetradesmus obliquus) and transformation-proficient (Phaeodactylum tricornutum, Microchloropsis gaditana, and Nannochloropsis oceanica) microalgae. C. sorokiniana rapidly degraded both linear and plasmid DNA, while T. obliquus exhibited intermediate activity. In contrast, no detectable degradation was observed in the transformation-proficient species. DNA degradation in C. sorokiniana occurred within minutes, and nuclease activity in both recalcitrant species was inhibited by EDTA and Zn2+, consistent with a metal ion-dependent endonuclease. These findings suggest that intracellular nuclease activity may represent an underexplored barrier to exogenous DNA stability during transformation and provide a potential target for improving genetic transformation of recalcitrant microalgae.

Oncogenic mutations are known to occur at non-random rates and specific hotspots, yet the structural and thermodynamic factors underlying these hotspots remain poorly understood. This study presents molecular dynamics simulations of 22 cancer hotspots across 10 oncogene families, simulated as histone-DNA complexes. Across 18 of 22 mutations, thermodynamic effects were predominantly localized to within 6 [A] of the DNA-histone interface (mean capture 104%), with van der Waals interactions driving the effect at the thermodynamic extremes, indicating that oncogenic mutations alter nucleosome binding through precise local contact changes rather than global structural rearrangements. Further, analysis revealed a gene-family correlated pattern in thermodynamic stability. RAS family mutations showed a consistent trend toward nucleosome stabilization (mean {Delta}{Delta}G = -4.82 kcal mol-1), while kinase domain mutations trended toward destabilization (mean {Delta}{Delta}G = +40.12 kcal mol-1), a difference reaching statistical significance in this exploratory analysis (Mann-Whitney U, p = 0.008). This interface-specific mechanism, combined with the gene-family-correlated thermodynamic pattern, provides a biophysical framework for understanding nucleosome-level contributions to cancer hotspot mutation biology.

DNA polymerases generally interact with the processivity clamp (DnaN/{beta}-clamp) during replication via clamp-binding motifs (CBMs). Since a CBM was not detected in DNA Polymerase I (PolI) from E. coli, it is generally believed that PolI performs its functions in genome replication and repair, independently of the {beta}-clamp. We observe that, while PolI and {beta}-clamp proteins from E. coli do not interact, their homologs from Mycobacterium tuberculosis bind strongly, and the binding site localises to the Klenow fragment of PolI. The cryo-EM structure (4.9 [A]) of the Mtb-PolI-Klenow: Mtb-{beta}-clamp complex, coupled with continuous heterogeneity analysis of the cryo-EM data and binding experiments, showed that the primary CBM in PolI may be defined by the stretch 457QLSLLDD463. The crystal structure (2.9 [A]) of the {beta}-clamp with the CBM peptide revealed specific interactions that stabilise the peptide within the {beta}-clamp's canonical binding site. Phylogenetic analyses across different bacteria showed that the identified CBM is present only in PolI from members of the order Mycobacteriales. Overall, the interaction between PolI with the {beta}-clamp, mediated by a CBM unique to the Mycobacteriales order, highlights the mechanistic distinctiveness of genome replication in Mtb and phylogenetically related organisms, and has important implications for drug discovery.

Background. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that provides an alternative route for electron transfer, contributing to respiratory flexibility and redox homeostasis under stress conditions. In trypanosomatids, AOX displays a highly uneven distribution associated with extensive diversification of mitochondrial electron transport chains (ETCs). In addition to canonical AOX, many trypanosomatids encode an enigmatic AOX-like protein (AOX-L), whose evolutionary origin and functional significance remain unresolved. Results. Here, we investigated the evolutionary distribution, phylogenetic relationships, structural conservation, and functional divergence of AOX and AOX-L across euglenozoans. Comparative genomic analyses revealed a patchy distribution of both proteins among kinetoplastids, with multiple independent losses during evolution. Phylogenetic analyses demonstrated that AOX and AOX-L represent distinct evolutionary lineages, indicating independent origins rather than duplication-derived divergence. While AOX showed a conserved eukaryotic origin, AOX-L occupied a separate phylogenetic position and lacked key residues required for ubiquinol oxidation. Structural modelling revealed that AOX-L retained the characteristic AOX fold, predicted membrane association, and dimeric organization, but lacked the catalytic architecture necessary for enzymatic activity. Transcriptomic and biochemical analyses of four AOX-containing trypanosomatid species showed that AOX expression and activity correlated with mitochondrial ETC organization. Species lacking cytochrome-dependent complexes III and IV displayed substantially higher AOX expression and enzymatic activity, consistent with AOX functioning as the primary terminal oxidase in these lineages. Conversely, species retaining a canonical ETC exhibited lower AOX activity, suggesting a role in metabolic flexibility and redox regulation. Conclusions. Our study provides a comprehensive evolutionary and functional framework for alternative oxidases in trypanosomatids. We demonstrate that AOX and AOX-L are evolutionarily distinct proteins with different predicted functions: AOX maintains respiratory electron flow according to lineage-specific mitochondrial requirements, whereas AOX-L represents a structurally conserved but catalytically inactive protein family that may have acquired an alternative regulatory role. These findings highlight how mitochondrial respiratory components diversify during eukaryotic evolution and provide a basis for future investigations into the biological function of AOX-L.

In eukaryotic cells, the positive transcription elongation factor b (P-TEFb) plays a critical role in the transition of RNAPII from the paused state to actively transcribing mode. Although three different cyclin (Cyc) T (CycT1, T2a, and T2b) interact with CDK9 to form functional P-TEFb, a majority of P-TEFb complexes are comprised of the CycT1 and CDK9. To analyze the role of the CycT1 subunit of P-TEFb on cellular transcription, we established HEK293T cells lacking CycT1 proteins (CycT1-KO cells). While no apparent growth defects were observed with CYcT1-KO cells, the level of CDK9 was decreased and no compensatory over-expression of CycT2 was observed, suggesting that the reduced level of functional P-TEFb complexes is sufficient for the normal cell growth. Reporter gene assays indicate that NF-kappaB-dependent transcription induced by PMA was impaired in CycT1-KO cells although the function of NF-kappaB (P65) per se was not affected. On the other hand, AP1-dependent transactivation induced by PMA was unaffected in CycT1-KO cells. Moreover, transcription stimulated by JQ1, a strong P-TEFb inducer, was reduced in CycT1-KO cells. Data of biochemical analysis indicate that P-TEFb with CycT1 (P-TEFb (CDK9:CycT1)) was efficiently released from 7SKsnRNP by JQ1 while P-TEFb (CDK9:CycT2) was not affected by JQ1. Transcriptome analysis indicated that the lack of CycT1 had a minor effect on the steady-state transcription. However, expression of JQ1-dependent genes was severely reduced in CycT1-KO cells. Interestingly, referring to gene expression profiles of 33 different types of cancer revealed that 11 out of 13 top JQ1-dependent genes were also highly upregulated in Pancreatic Adenocarcinoma. From these results, we conclude that although many genes can be regulated by both P-TEFb (CDK9:CycT1) and P-TEFb (CDK9:CycT2), CycT1 plays a critical role in regulating highly inducible genes, which are also aberrantly regulated in a particular type of cancer.

Palmitate-associated lipotoxic stress activates the endoplasmic-reticulum stress sensor protein IRE1, but how IRE1 signaling influences genotoxic-stress responses in triple-negative breast cancer remains unclear. Here, we investigated IRE1 and XBP1 signaling in MDA-MB-231 cells exposed to palmitate and etoposide. Under combined lipotoxic and genotoxic stress, XBP1 depletion increased DNA-damage accumulation in IRE1 wild-type cells but not in IRE1-deficient cells, whereas IRE1 re-expression reduced DNA damage. Pharmacologic IRE1 RNase inhibition also increased etoposide-associated DNA damage, supporting a protective role for IRE1 RNase-dependent signaling. IRE1 status and XBP1 manipulation differentially affected H2A.X and {gamma}H2A.X abundance, indicating context-dependent regulation of chromatin-associated DNA-damage signaling. At the RNA level, H2A.X was enriched in IRE1 immunoprecipitates, and the H2A.X-linked DNA-damage-response transcripts HUWE1 and BRCA2 also associated with IRE1 under combined stress. These findings link palmitate-activated IRE1-XBP1 signaling to genome-protective responses during genotoxic stress and identify a network of IRE1-associated DNA-damage-response transcripts in triple-negative breast cancer cells. Direct IRE1-mediated cleavage of these transcripts remains to be established.

Bacterial insertion sequences (IS) are compact transposable elements that encode proteins required for their mobility and maintenance, yet many also encode accessory proteins with poorly understood functions. For example, IS605-family elements often encode a transposase called TnpA and an RNA-guided nuclease called TnpB that supports transposon maintenance, alongside an additional ribbon-helix-helix protein named Arc. Though the roles of TnpA and TnpB have been extensively studied in recent years, the enigmatic function of Arc has not been investigated. Here, we show that Arc acts as a transcriptional repressor to directly bind the transposon's native promoter sequence regulating TnpA and TnpB gene expression. By systematically testing Arc-containing IS605 elements, we identified a conserved binding pattern at intergenic transposon sequences neighboring protein-coding genes through chromatin immunoprecipitation and sequencing analyses. We then used fluorescence reporter assays and demonstrated that these intergenic sequences function as strong promoters, and that the presence of Arc dramatically reduces their gene expression. Together, these findings identify Arc as a transposon-encoded transcriptional repressor, revealing a regulatory layer that may promote long-term persistence of IS605-family elements by keeping their activity in check. The widespread association of Arc homologs with diverse mobile elements and cellular genes suggests that these compact regulators may more broadly restrain the expression of neighboring genetic machinery across varied genomic contexts. neighboring genetic machinery across varied genomic contexts.

The intrinsically disordered tardigrade Dsup protein has garnered attention for its ability to protect cells and organisms from radiation and oxidative stress. Our lab has previously described Dsups protective effect in yeast, finding that, while Dsup expression comes with a fitness cost, it improves survival under many stressors, including the heavy metal cadmium. While Dsups protection against oxidative stress and radiation is well-established, the mechanism by which it protects against other stressors is underinvestigated. To address this knowledge gap, we screened a collection of 4000 yeast deletion mutants expressing Dsup in the presence of three doses of cadmium to identify synergistic/synthetically lethal genes and processes. We find that Dsup broadly buffers many processes/genes at lower levels of cadmium stress but at higher doses, cells shift to buffering specific genes/processes. These results identify host genetic processes that influence Dsups ability to confer cadmium resistance to organisms.

Kaposi's sarcoma (KS) is an angioproliferative tumor associated with infection by human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV). Among the viral genes involved in KS pathogenesis, the viral G protein-coupled receptor (vGPCR) plays a key role in oncogenic transformation and angiogenesis, and its sustained activity contributes to tumor development and maintenance. Quercetin (QUE), a naturally occurring flavonoid with well-recognized anticancer properties, has been shown to reduce cell viability and proliferation and to induce apoptosis in vGPCR-transformed endothelial cells. This study further investigated the mechanisms underlying the antineoplastic effects of QUE in vGPCR cells, an in vitro model of Kaposi's sarcoma. Initially, QUE promoted the activation of MAPK signaling pathways in vGPCR cells, increasing the phosphorylation levels of ERK1/2, JNK1/2, and p38, without affecting AKT phosphorylation. In parallel, QUE increased intracellular oxidant levels, followed by lipid peroxidation and DNA damage, supporting a link between oxidative imbalance and genotoxicity. However, increased total glutathione content, and unaltered Gpx4 mRNA expression, suggest the induction of oxidative stress and compensatory antioxidant responses. Furthermore, increased intracellular oxidant levels were associated with MAPK signaling activation, particularly JNK1/2, whereas ERK1/2 and p38 activation may reflect a compensatory response that is insufficient to overcome QUE-induced cytotoxicity. Taken together, these findings indicate that QUE exerts antiproliferative effects in vGPCR cells through a mechanism involving oxidative stress, MAPK activation, and DNA damage, supporting its potential relevance in the study of therapeutic strategies for virus-associated malignancies.

Telomeres, the ends of linear chromosomes, form a cap composed of specific telomeric DNA associated with specialized proteins that ensures efficient protection against chromosome degradation and, consequently, overall genome instability. Three main complexes have been implicated in chromosome end protection, namely telomerase, shelterin, and the CST complex (CTC1/Cdc13-STN1-TEN1). In a recent study, we uncovered mutants of the S. cerevisiae CST in which damage was sensed both by the two major spindle checkpoints and the major DNA damage checkpoint. In the best-studied of these mutants, stn1-sz2, the stability and organization of the mitotic tubulin spindle was profoundly affected, as were the organization of the centrosomes and the kinetochores. Here, we find that the stn1-sz2 mutant is hypersensitive to deletion mutants of two members of the kinetochores, Ctf19 and Mcm21, which correlated with mis-localization of their associated subunit in the COMA complex, Okp1, during anaphase. Defects in interactions between the kinetochores and the tubulin spindle are entirely sensed by the SAC (spindle assembly checkpoint). Here, we find that some of the stn1-sz2 damage is recognized by the Mad2 checkpoint protein independently from the SAC. The data presented here contribute to a better understanding of the damage in stn1-sz2, the prototype of novel mutants of telomeric CST characterized by mitotic-spindle-damage in addition to the classical telomeric-DNA-damage.

The base excision repair pathway removes small base lesions from DNA and is initiated in humans by one of eleven DNA glycosylases with overlapping substrate specificities. Despite their central role in genome maintenance and transcription, systematic datasets describing small-molecule binders or inhibitors of DNA glycosylases are lacking. Here, through a collaborative effort across Scandinavia, we establish a high-throughput biochemical assay platform for human DNA glycosylases and use it to generate a systematic small-molecule screening dataset. A chemogenomic library was screened against nine glycosylases with assays of sufficient quality for hit identification, yielding primary screening and concentration-response data and identifying multiple previously unreported inhibitors. All primary and processed data, along with detailed assay protocols and metadata, are publicly available to support reuse in chemical biology, further compound optimization, assay development and comparative studies of DNA repair enzymes.

Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor. In vitro enzymatic assays showed that KPB82 inhibited ACER2 with an IC50 of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC50> 100 {micro}M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn2+-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.

Ultraviolet-C (UV-C) radiation is a potent environmental stressor that induces DNA damage, accumulation of reactive oxygen species (ROS), and cellular dysfunction. This also initiates coordinated molecular responses to regulate cellular adaptation and recovery. Small extracellular vesicles (sEVs) have emerged as key mediators of intercellular communication through the selective incorporation of microRNAs (miRNAs), proteins, and lipids. However, the temporal changes of sEV-associated cargo during the progression from UV-C-induced damage to recovery remain uncharacterized. We hypothesised that UV-C exposure induces stage-specific changes in sEV cargo (miRNA and proteins) that promote the transition of cells from damage to the response and recovery phases. To examine this, a time-resolved UV-C exposure model was established using Caenorhabditis elegans (C. elegans). In this paper, we studied 2.5 hours of UV-C exposure, including cellular damage, intracellular ROS levels, and DNA integrity, to define the damage and repair state. Based on these temporal variations, samples were collected: i) immediately after exposure, ii) 6 hours post-exposure, and iii) 16 hours post-exposure were designated as damage, response, and recovery conditions, respectively. sEVs isolated from whole-worm preparations were characterised using biophysical and molecular approaches, followed by miRNA sequencing and proteomic analyses. Integrative analysis further identified miRNA-protein associations and associated them with pathways involved in stress adaptation, metabolism, signal transduction, and cellular homeostasis. Collectively, these findings provide a temporal molecular framework of sEV cargo associated with UV-C-induced damage, response, and recovery in C. elegans. Overall, this study identified candidate molecular signatures and miRNA-protein associations that underlie the temporally selective packaging of sEV-associated cargo following UV-C-induced damage, providing a basis for future tissue-specific and mechanistic investigations.

Acute radiation syndrome remains a major consequence of high-dose ionizing radiation exposure, and effective pharmacological countermeasures are limited. Nitro-fatty acids possess anti-inflammatory and cytoprotective properties, suggesting potential effectiveness in radiation injury. Here, we investigated the radioprotective effects of 10-nitro-oleic acid (10-NO2OA) in mouse models of total body irradiation (TBI). C57BL/6 mice were exposed to either sublethal (4 Gy) or LD50-level (7.5 Gy) gamma irradiation and treated with 10-NO2OA according to pre-irradiation, post-irradiation, or combined treatment regimens. Survival, hematological responses, bone marrow cells (BMCs)/colony-forming units (CFU), plasma granulocyte colony-stimulating factor (G-CSF), NRF2-responsive signaling, and intestinal responses were evaluated. Administration of 10-NO2OA before and after irradiation significantly improved 30-day survival following 7.5 Gy TBI (57% vs. 20% in vehicle-treated controls). In contrast, post-irradiation treatment alone was ineffective, whereas pre-irradiation treatment alone produced a non-significant trend toward improved survival. The protective effect was absent in BALB/c mice, indicating strain dependence. Although 10-NO2OA did not prevent long-term radiation-induced cytopenia, it increased neutrophil and platelet counts, elevated plasma G-CSF levels shortly after irradiation, and transiently enhanced BMC counts. In irradiated BMCs, 10-NO2OA activated NRF2-responsive gene expression in vitro and in vivo. Exploratory intestinal analyses revealed improved macroscopic appearance and partial restoration of irradiation-suppressed cytokine and growth factor production. In conclusion, 10-NO2OA improves survival following LD50-level gamma irradiation and is associated with modulation of hematopoietic, cytoprotective, and intestinal responses. These findings identify 10-NO2OA as a promising candidate radioprotective agent and support a multifactorial mechanism of action.

Minor introns are rare but evolutionarily conserved introns in the human genome. Defects in minor spliceosome components have been reported to cause several human diseases. Mutations in ZRSR2 are frequently observed in myelodysplastic syndrome (MDS) patients. However, the precise molecular mechanism by which these mutations cause a loss of ZRSR2 function in minor intron splicing remain elusive. In this study, we characterized the impact of MDS-causing missense mutations in ZRSR2. Most of the mutant ZRSR2 proteins we tested showed different subnuclear localization pattern from that of wild-type ZRSR2 protein. Co-immunoprecipitation (co-IP) analysis revealed that UHM missense mutations disrupted the interaction between ZRSR2 and SF3B complex. We also demonstrated that ZRSR2 preferentially immunoprecipitated minor spliceosome components over major spliceosome components and it directly interacts with SF3B1. Furthermore, co-IP analysis utilizing a variety of ZRSR2 mutants revealed that mutations in the UHM and Zn2 domains significantly attenuated the interaction with SF3B complex. Finally, UHM and Zn2 mutations markedly disrupted the supporting activity of ZRSR2 for minor intron splicing. Our results indicate that disruption of the physical interaction with SF3B complex is one of the hallmarks of MDS-causing mutations of ZRSR2, which results in aberrant splicing of minor introns.

Pathogenic ERCC2 variants cause xeroderma pigmentosum (XP), trichothiodystrophy (TTD) or both, yet variant effect scores are usually interpreted only as measures of pathogenicity rather than of which disease mechanism is disrupted. XPD, the ERCC2-encoded TFIIH subunit, functions in both nucleotide excision repair and transcription. Using yeast complementation deep mutational scanning, we measured the effects of nearly all XPD amino acid substitutions. The assay was mechanism-selective: it preferentially reported transcription-associated function, with pronounced intolerance at the p44 interface, whereas many substitutions affecting DNA binding and helicase activity retained near-wild-type fitness. Accordingly, TTD variants had much lower fitness than XP variants. Computational predictors discriminated pathogenic from benign variants similarly across phenotypes, but the DMS distinguished XP from TTD variants better than all 73 predictors tested. Phenotype-specific ACMG/AMP calibration provided evidence in both directions for TTD but mainly pathogenic evidence for XP. Thus, the selectivity of functional assays, often viewed as a limitation, can reveal disease mechanisms and support phenotype-aware variant interpretation.

Many RNA-binding proteins play pleiotropic roles in the cell, often through disparate domains that can be decoupled. Here we undertook a mutational analysis of yeast RNA-binding protein Ssd1 to better understand domains and processes required for tolerating extra chromosomes, a special class of aneuploidy. Ssd1 is important for cellular fitness when cells carry extra chromosomes, for reasons that are not clear. Ssd1 has multiple distinct domains, many of which are poorly characterized, including multiple RNA binding folds, nuclear import and export signals, an intrinsically disordered domain, an RNA polymerase II-interacting region, and many phosphorylation sites. We measured multiple phenotypes, including the ability to tolerate an extra copy of yeast chromosome XII as a representative aneuploid. Mutating RNA binding domains disrupts fitness in aneuploid, but not euploid, cells and ablates binding of a representative Ssd1 target. However, mutations affecting phase separation or interaction with the kinase Cbk1, which regulates Ssd1 granule formation during the cell cycle, did not affect aneuploidy tolerance. Evaluating correlations across these and other growth phenotypes informed on functional relationships among domains. We discuss implications for Ssd1 function and its role in aneuploidy tolerance in yeast.

Background: Protein-coding DNA exhibits characteristic three-base periodicity. In unaligned DNA libraries, however, this signal should cancel if fragment boundaries are uniformly distributed across codon phases. Yet, we find that in multiple metagenomic libraries, base frequencies depended systematically on distance from fragment boundaries, producing a period-3 pattern. We investigated the origin of this unexpected signal. Results: The signal was observed in modern metagenomic datasets and widely across ancient-DNA datasets. We developed a new alignment-free algorithm to infer latent triplet-phase structure from nucleotide composition. The inferred shifts were strongly nonuniform at fragment boundaries, explaining the read-coordinate periodicity. We initially suspected a protocol artifact. To test an alternative, we derived a probabilistic framework linking nucleotide-dependent boundary selection to codon-phase frequencies. The framework showed that, because nucleotide composition differs among codon positions, preferential breakage at particular nucleotides biases the phases represented at fragment boundaries. Simulations confirmed this mechanism: purine-associated fragmentation produced strong period-3 coherence in a coding-dense bacterial genome, whereas no comparable genome-wide effect appeared in the human reference genome. Conclusions: The observed read-coordinate three-base periodicity does not require an intrinsic phase bias in the source DNA. Instead, sequence-dependent fragment formation or recovery can couple boundaries to codon phase. Purine-associated post-mortem fragmentation plausibly explains the widespread phenomenon in ancient-DNA libraries, although PCR- or library-specific mechanisms may contribute to it in modern datasets. Our alignment-free algorithm provides a practical method for estimating and normalizing phase shifts before searching for positional nucleotide patterns, preventing boundary-associated bias from being mistaken for an intrinsic biological pattern.