DNA Repair

Previous studies revealed a crosstalk between intronic polyadenylation (IPA) and the DNA damage response (DDR). Indeed, genotoxic agents, including radiations and anticancer drugs (e.g., cisplatin that crosslinks DNA), regulate the ratio of IPA to last-exon transcripts in many genes. Conversely, multiple genes involved in the DDR, especially homologous recombination, are regulated at the IPA level. The U1 small nuclear RNA (snRNA) widely represses IPA, thereby enhancing full-length gene transcription. However, besides its implication in IPA regulation by ultraviolet-C radiation, little is known about U1 snRNA effects on the DDR and on cell sensitivity to genotoxic agents. Here, we show that U1 snRNA blockade using an antisense oligonucleotide (U1-AMO) in lung cancer cell lines enhances cell growth inhibition by cisplatin, through an increase in cisplatin-induced DNA damage. 3-seq analysis indicates that U1 snRNA blockade represses full-length mRNA expression of multiple genes of the nucleotide-excision repair and Fanconi anemia pathways, which are involved in the repair of cisplatin-DNA crosslinks. Our 3-seq analyses also reveal that moderate doses of U1-AMO and cisplatin upregulate the IPA:LE isoform ratio in overlapping but distinct sets of genes, and that U1-AMO prevents cisplatin effects on the IPA:LE ratio in a large subset of genes. Altogether, these data extend the crosstalk between IPA and the DDR and suggest that U1 snRNA targeting may be used to sensitize cancer cells to genotoxic agents.

PARK7 (DJ-1) is a redox-sensitive stress-response protein that supports cellular adaptation, but its role in post-transcriptional responses to genotoxic stress remains unclear. We investigated whether DJ-1 abundance determines the miRNA response to X-ray-induced DNA damage. Integrated mRNA-seq and small RNA-seq across DJ-1 states in HEK293 cells revealed a striking divergence in miRNA plasticity. DJ-1 depletion by siRNA produced minimal miRNA remodeling, with only 34 (6.3%) miRNAs differentially expressed after irradiation. In contrast, elevated DJ-1 markedly increased miRNA plasticity: irradiation altered [~]37% of detectable miRNAs, accounting for [~]90% of miRNA reads, and extensively redistributed the miRNA pool. DJ-1 overexpression was also associated with remodeling of the miRNA regulatory machinery, particularly components involved in miRNA sorting and stability, suggesting feedback regulation of the miRNA pool. Comparison of precursor and mature species revealed substantial uncoupling between transcription and mature miRNA abundance, implicating regulation at the levels of processing, maturation, or stability. Radiation-responsive coding genes in DJ-1-overexpressing cells were relatively depleted of miRNA binding sites, supporting preferential regulation of upstream regulatory nodes rather than the bulk transcriptome. Together, these findings identify DJ-1 as a determinant of post-transcriptional signaling plasticity, enabling dynamic remodeling of the miRNA regulatory state in response to genotoxic stress. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/744036v1_ufig1.gif" ALT="Figure 1000"> View larger version (38K): org.highwire.dtl.DTLVardef@18824aorg.highwire.dtl.DTLVardef@111d8bcorg.highwire.dtl.DTLVardef@ac33b4org.highwire.dtl.DTLVardef@17698d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging, with a life expectancy of less than two decades. HGPS is commonly caused by a point mutation in the LMNA gene which codes for lamin A, a vital component of the nuclear lamina. The HGPS mutation activates a cryptic splice site and leads to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also produced in small amounts in healthy individuals and has been implicated in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs), and alterations in DSB repair. DSB repair in mammalian cells normally occurs by either homologous recombination (HR), an accurate, templated form of repair, or by DNA end-joining (EJ), a non-templated rejoining of DNA ends. EJ is error-prone, although a portion of EJ events occurs precisely with no alteration to joined sequences. Previously, we reported that over-expression of progerin increased EJ relative to HR and decreased the precision of EJ. In our current work, we designed a novel model experimental system using derivatives of thymidine kinase (tk)-deficient mouse fibroblasts and incorporating a loss-of-function assay to further explore progerins impact on EJ. We established cell lines containing an integrated copy of a functional herpes tk gene with an embedded recognition site for endonuclease I-SceI. We examined EJ at the nucleotide level following induction of a DSB within the tk gene by expression of I-SceI and subsequent selection for cells that lost tk gene function. Comparison of EJ products recovered from cells expressing progerin versus from cells not expressing progerin revealed that progerin expression provoked larger DNA deletions associated with DSB repair as well as recovery of multiple repair products from individual cells, suggesting progerin impedes re-joining of DNA ends.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Orc6 is the smallest and least evolutionarily conserved among all ORC subunits. In Drosophila, Orc6 binds tightly with the core ORC(1-5) and is required for DNA binding and replication initiation, whereas in Xenopus and human systems Orc6 loosely associates with the rest of the complex resulting in some differences for replication-associated activities. Despite these variations, Orc6 remains essential for viability in all species. In current study we analyzed specific residues within the C-terminal 11 helix that is critical for stable association of Orc6 with the ORC complex in Drosophila. Human Orc6 lacks these residues, however it possesses a strong nuclear localization signal (NLS) that is absent in Drosophilidae. We propose that this NLS drives human protein to the nucleus and compensates for weaker Orc6-ORC(1-5) interactions by increasing the nuclear concentration of Orc6 and shifting the equilibrium toward formation of the fully assembled ORC complex at the DNA.

Sliding clamp proteins play central roles in DNA metabolism, including replication and repair. The ring-shaped E. coli beta clamp accommodates double-stranded DNA and serves as a platform for proteins involved in multiple DNA transactions. The inner pore of the beta clamp harbors a series of positively charged and polar residues that can bind to the negatively charged backbone of the DNA. These residues are arrayed so that they do not align with the charged phosphates of the DNA backbone. It is hypothesized that this arrangement of these residues provides for the movement of the clamp on DNA as it alternates which residues are bound to the DNA backbone. In this work, we mutated specific charged and polar residues that project into the inner pore of the beta clamp. The beta clamp variants are dimers and have similar thermal stability and in general a similar ability to complement a temperature sensitive strain for growth. One exception was beta-Q149A, which appeared as higher-order species on a native gel although its hydrogen-deuterium exchange pattern measured by mass spectrometry was overall similar to WT beta. These variants all had decreased binding to DNA after loading. Optical tweezers experiments were used to monitor loading on single DNA molecules and measure the rate of beta clamp sliding on DNA. Consistent with the hypothesized role of positively charged residues in the beta inner pore, mutation of one residue resulted in a faster rate of sliding on DNA.

During replication, nucleolytic processing of apurinic/apyrimidinic (AP) sites in single-stranded (ss)DNA is attenuated by the evolutionarily conserved 5-hydroxymethylcytosine binding, embryonic-specific (HMCES) protein. HMCES forms a covalent thiazolidine linkage with the ring-opened aldehyde form of a ssDNA AP site to stabilize the AP site and suppress the formation of DNA double-strand breaks. The resulting HMCES DNA-protein cross-link (DPC) can then be digested by the SPRTN protease and bypassed through mutagenic translesion synthesis (TLS). Here, we use Xenopus egg extracts and molecular dynamics simulations to investigate how HMCES-DPC formation influences the mutagenicity of AP site bypass. We show that SPRTN processes the HMCES-DPC to a five amino acid peptide adduct prior to TLS. Surprisingly, the mutagenicity of HMCES-DPC bypass is insensitive to the extent of DPC proteolysis and depends only on cross-link formation, which traps the AP site in a more dynamic ring-opened configuration. We further show that the spectrum of mutations produced during bypass of HMCES-adducts strongly depends on the template strand nucleotide immediately 5 of the AP site. Our data support a model in which HMCES-DPC formation increases the conformational flexibility of the DNA template, allowing template slippage and use of the 5 template nucleotide to direct insertion opposite the adducted AP site.

Blue light using Flavin (BLUF) proteins are microbial photoreceptors that are involved in various physiological responses. Their occurrence and biochemical properties in fungi remain poorly understood. Here, we investigated a putative BLUF photoreceptor from the corn-smut fungus Mycosarcoma maydis (MmBLUF). Domain analysis, multiple sequence alignment of BLUF core regions, and structural modelling indicated conserved canonical BLUF fold and flavin-pocket residues. However, when heterologously expressed, UV-visible and fluorescence spectroscopy revealed different spectral behaviour than canonical BLUF protein. Further, we tested the role of extended N-terminus in modulation of chromophore binding by expressing N-terminus truncated protein variants. Our results suggest that the unusual spectral behaviour is not linked to the truncation construct (extended N-terminus), which also showed similar spectral features, indicating that the extended N-terminus is unlikely to account for an unusual photodynamics characteristics. Our findings support MmBLUF as a structurally conserved putative fungal BLUF-like photoreceptor with different photochemical properties. Further studies are required to establish its chromophore identity, photocycle and function of this unusual BLUF-like domain from fungal system.

Oxidative DNA damage is a common threat to genomic integrity, arising from endogenous metabolic processes and environmental exposures. If unrepaired, such oxidative DNA damage promotes mutagenesis and genomic instability. Cells counter this through base excision repair (BER), a multi-step pathway requiring the coordinated action of several proteins. Central to BER, DNA polymerase beta (pol ) locates single-nucleotide (1-nt) gaps and inserts the correct nucleotide, while x-ray repair cross-complementing 1 (XRCC1) is a scaffold protein that forms a stable complex with pol to coordinate BER factors at DNA damage. XRCC1 enhances BER efficiency, though the mechanism by which this occurs is unclear. Pol {beta} is proposed to be recruited to DNA damage by undamaged DNA scanning interactions, but this behavior has not yet been directly observed. Additionally, the influence of other BER proteins on pol recruitment, particularly XRCC1, remains unclear. Here, we used correlative optical tweezers-fluorescence microscopy to visualize DNA search and damage recognition by pol and XRCC1. We characterize each factor individually, examine their behavior as the pol -XRCC1 complex, and assess their interplay with apurinic/apyrimidinic endonuclease 1 (APE1), the enzyme upstream of pol in BER. We find that pol locates damage through 3D-diffusion, whereas XRCC1 exhibits both 3D- and 1D-diffusion. In combination, XRCC1 dramatically shifts pol {beta} search towards 1D-diffusion, enabling interrogation of non-damaged DNA using both search mechanisms. When both APE1 and pol are present, the pol -1nt gap complex is highly stable, with APE1 largely unable to disrupt the damage-bound pol . Together, these findings demonstrate that XRCC1 reshapes pol {beta} search behavior to promote efficient local damage recognition, providing a mechanistic basis for how BER factors coordinate lesion detection and processing to maintain genomic stability.

Exogenous and endogenous mutagens generate a wide variety of DNA lesions, including bulky adducts, chemical modifications, and single- or double-stranded breaks. A phenomenon called "lesion segregation," in which lesions evade repair and persist for multiple cell divisions, has recently been documented in tumors and healthy somatic tissues from mice and humans, respectively. Persistent lesions can generate multi-allelic variants (MAVs) by serving as templates for multiple rounds of error-prone replication. By reanalyzing data from a large C. elegans mutagenesis experiment, we observed robust evidence for MAVs at a small fraction (~0.2%) of mutated sites in the offspring of strains treated with alkylating agents. Because these sequencing data were derived from the progeny of a single F1 animal -- itself the offspring of a mutagenized P0 -- all mutations should be biallelic. The presence of multi-allelic variation implies that some DNA lesions are transmitted to the F1 zygote, evade repair, and are repeatedly bypassed by error-prone polymerases during embryogenesis. We suspect that many more lesions are inherited than is suggested by MAV prevalence, and that a large fraction of biallelic mutations are also caused by inherited lesions. Our results demonstrate that DNA lesions serve as durable, transgenerational templates for mutagenesis in C. elegans . We speculate that lesion segregation in the early embryo may be a source of mosaicism and genetic diversity in humans, as well.

Mutations in mitochondrial DNA (mtDNA) compromise ETC activity and impair oxidative phosphorylation. Since eukaryotic cells contain multiple copies of mtDNA, the resulting phenotype depends on the proportion of mutant mitochondrial genomes (the heteroplasmy level). Using isogenic cell lines carrying similar mtDNA deletions, a linear decline in cellular respiration was observed as mitochondrial DNA heteroplasmy increased. Despite this, cellular redox imbalance did not change until heteroplasmy exceeded 50%. As heteroplasmy increased past 70%, cells also exhibited an integrated stress response (ISR) and impaired translation was observed. These defects were reversed by either addition of asparagine or overexpression of pyruvate carboxylase (PC). The dependence on exogenous asparagine in other respiration-deficient cells was found to correlate inversely with the PC expression level. For example, patient-derived thyroid tumor cells, harboring high heteroplasmy for a Complex I mtDNA mutation and low levels of PC, exhibited asparagine auxotrophy, and L-asparaginase treatment suppressed tumor growth. Together, these findings demonstrate a role for mitochondrial pyruvate carboxylase in cellular asparagine synthesis under conditions of compromised respiratory activity.

BACH1 is a heme-regulated basic-leucine-zipper containing transcriptional repressor that binds its DNA recognition elements as a heterodimer with MAFK. Heme-binding is thought to be mediated by several Cys-Proline (CP) motifs and this results in dissociation of the heterodimer from DNA. The mechanism of heme-binding and heme-mediated DNA dissociation remains unresolved. We have used UV-visible spectroscopy, 2D-NMR and DNA-binding assays to explore both heme-binding and DNA dissociation of a minimal BACH1 construct containing 2 CP motifs (C492(CP5) and C646(CP6)) flanking the DNA-binding domain. We find that heme is able to bind to both CP motifs, but also to other non-CP cysteines and histidines in the construct. Using NMR spectroscopy, we identify a structured binding pocket in which heme interacts with both C646(CP6) and Cys621. However, DNA-binding assays show that C646(CP6) is not required for heme-mediated DNA dissociation of the BACH1:MAFK heterodimer. Using UV-visible spectroscopy we show that C492(CP5) also recruits heme with a second ligand, a conserved histidine, His559, in the BACH1 DNA-recognition helix. Mutation of C492(CP5) reduces but does not abolish heme-mediated dissociation from DNA. Our findings suggest a mechanism for heme-binding to BACH1 and heme-mediated dissociation from DNA.

We present a simple and efficient way to add cis dialdehydes, phosphoryl groups, or cis diols to the 3 prime end of oligonucleotides using periodate oxidized nucleotides (oNTPs) and terminal deoxynucleotidyl transferase (TdT). The 3 prime end cis dialdehyde-modified oligos are generated by incubating TdT with an oligo for several minutes followed by addition of a oNTP and incubated at 30 degrees C for 30 minutes to an hour. After allowing the addition of the cis dialdehydes, heating the reaction mixture at 90 to 95 degrees C for 10 minutes yields oligonucleotides with 3 prime phosphoryl groups. The 3 prime cis diol modified oligos are synthesized by starting with 3 prime cis diol nucleotides (HO-NTPs). The cis dialdehyde modified oligonucleotides and cis diols may then be used for a variety of investigations such as studying the interaction of proteins with the 3 prime end of DNA, and possibly RNA. As an example, we demonstrate the efficacy of using an oligonucleotide modified with oGMP at the 3 prime end as an affinity label for TdT and identified a peptide fragment that has been shown to contain two of three aspartate residues found to be in the TdT active site.

Mutations in p53 and its 12 isoforms can alter its functions. As N-terminally truncated isoforms of p53 (delta40p53, delta133p53, and delta160p53) participate in tetramer formation, they are important regulators of cancer fate. Although delta40p53- and delta133p53-mediated regulation of cancer is well reported, the mechanism underlying delta160p53 production and its functional role remains unclear. We investigated the internal ribosomal entry site (IRES)-mediated translation of {Delta}160p53 and its role in cancer regulation. As differential synthesis of delta160p53 was observed under different stress conditions, IRES-mediated translation of this isoform was demonstrated using bicistronic luciferase constructs. No cryptic promoters or splicing sites were detected in the IRES sequence. Cell death and late apoptosis were significantly decreased, while proliferation, the number of cells in the S phase, and drug resistance were induced by delta160p53. Furthermore, delta160p53 did not induce p53-responsive promoters. RNA sequencing analysis of delta160p53 overexpression showed similar results, along with the inhibition of other tumor suppressor genes. Overall, our results provide insights into IRES-mediated translation of delta160p53, which can be considered a novel target for cancer treatment.

Ubp3 is one of about 20 deubiquitinases (DUBs) in S. cerevisiae. The current view generally assumes that Ubp3 requires its interaction partner Bre5, which is proposed to function as a positive regulator. Accordingly, the Ubp3/Bre5 complex has been implicated in a broad range of cellular processes for example trafficking between ER and Golgi, stress granule formation and selective autophagy. However, the molecular basis of this proposed Bre5-dependent activity remains unclear. To address this at a molecular level, Ubp3, Bre5, and related constructs were heterologously expressed in E. coli, purified to homogeneity, and characterized in vitro. Both proteins contain folded domains as well as extensive intrinsically disordered regions (IDRs). Despite this structural complexity, the Ubp3/Bre5 complex could be isolated following either co-expression in vivo or after in vitro assembly. Unexpectedly, complex formation with Bre5 was not required for the catalytic activity of full length Ubp3. Furthermore, even the isolated catalytic domain of Ubp3 was fully active against two distinct substrates in the absence of Bre5, demonstrating that its deubiquitinating activity is intrinsically independent of Bre5. These findings indicate that the catalytic domain alone is sufficient for substrate cleavage, whereas the extensive IDRs of Ubp3 and its cofactor Bre5 might contribute to substrate recognition or specificity. Overall, this study challenges the prevailing model of Bre5-dependent activation of Ubp3 and provides new insights into the molecular organization of the Ubp3/Bre5 system. More broadly, it highlights the importance of intrinsically disordered regions in regulating deubiquitinase function and cellular signaling networks.

HP1 proteins are highly evolutionarily conserved chromatin-associated factors known to play essential roles in genome stability and nuclear organization. In mammals, three HP1 isoforms, HP1, HP1{beta} and HP1{gamma}, have been described, but their individual functions remain incompletely characterized. Here, we inactivated HP1 or HP1{beta} in different cell lines and quantified chromosomal breaks on metaphase spreads in the presence or absence of aphidicolin-induced replication stress. Loss of HP1, but not of HP1{beta}, led to a significant increase of chromosomal breaks on chromosome arms and within pericentromeric heterochromatin under these conditions. Mechanistically, loss of HP1 was associated with a reduction in replication fork velocity, suggesting that HP1 deficiency induces a replication stress that sensitizes specific genomic loci to replication perturbation. Consistent with this, HP1 loss was associated with a moderate but consistent increase in {gamma}H2AX and 53BP1 foci, an increased occurrence of DNA synthesis during mitosis, and enhanced recruitment of FANCD2, all recognized as hallmarks of common fragile site (CFS) expression. In addition, rescue experiments using a chromodomain mutant HP1 (V22M) unable to bind H3K9me3 indicated that HP1 protective function over these specific foci did not require its interaction with this histone mark. Altogether, these data indicate that, independently of its binding to H3K9me3, HP1 stabilizes specific genomic regions that behave as HP1-dependent fragile sites, at least in part by regulating replication fork progression, limiting mitotic DNA synthesis possibly by competing with FANCD2 for chromatin access at these regions.

Purpose Radiation injury to growth plates commonly leads to skeletal late complications including short stature, limb length-discrepancy, and scoliosis/kyphosis in pediatric oncology patients. We aimed to understand the acute responses of direct growth plate irradiation that result in skeletal late complications. Materials and methods We first established an in vivo model of focal growth plate irradiation that recapitulates the clinical development of skeletal late complications and used it to explore the responses of growth plate chondrocytes within the first 72 hours of radiation exposure. To monitor acute effects of radiation exposure on human chondrocytes, rare human growth plate biopsies were exposed to ionizing radiation ex vivo. Using these approaches, we applied clonal genetic tracing and immunofluorescence to monitor changes at the cellular and molecular levels. Functional in vivo perturbations were conducted with clinically-relevant autophagy inhibitor, hydroxychloroquine. Results Growth plate irradiation disrupted the continuous production of chondrocytes required for bone elongation and was associated with DNA damage throughout the growth plate. Indicators of growth plate activity, SOX9 and the phosphorylated form of ribosomal protein S6, decreased during a 6- and 24-hour post-irradiation window but returned to normal levels 72 hours after irradiation. We identified a surge in autophagic flux throughout the growth plate during this window, based on temporal SQSTM1 and LAMP1 protein levels. The earliest stages of these response mechanisms are conserved between species and relevant to humans. Hydroxychloroquine treatment immediately after radiation injury in mice impaired growth plate regeneration, resulting in more severe late complications. Conclusion Our findings demonstrate that autophagy is an important acute response to irradiation in growth plate chondrocytes, revealing a novel potential therapeutic target for preventing radiation-induced skeletal late complications.

Most eukaryotic mRNAs encode a single functional polypeptide. Following translation termination, both the large and small ribosomal subunits are typically released from the mRNA by ribosome recycling factors. However, after translating short upstream open reading frames (uORFs) within the 5 untranslated regions (UTRs), ribosomes can remain associated with the mRNA and reinitiate translation. This process is regulated by the heterodimer MCTS1*DENR (Tma20p*Tma22p in yeast). DENR/Tma22p harbors a SUI1 domain, structurally homologous to the translation initiation factor eIF1/Sui1p, which features a conserved, positively charged {beta}-hairpin loop critical for eIF1 function. Despite this structural similarity, the functional significance of specific elements within DENR/Tma22p remains unexplored. Here, we used in vivo reporter assays in Saccharomyces cerevisiae to quantify reinitiation efficiency following translation of either a short uORF (in the 5 UTR) or a full-length coding sequence (in the 3 UTR). Systematic analysis of single, double, and triple deletions of TMA20, TMA22, and TMA64 (a homolog of Tma20p*Tma22p) revealed that the Tma20p*Tma22p complex exerts a dominant role over Tma64p in modulating reinitiation, while exhibiting functional interplay between the two factors. Using knockout strains complemented with Tma22p variants, we further demonstrated that the positively charged residues of the {beta}-hairpin loop 1 are essential for Tma22p recycling activity. Unexpectedly, deletion of the entire SUI1 domain was less deleterious, and eIF1/Sui1p was able to partially substitute for the SUI1 domain of Tma22p within a chimeric protein context. Our findings establish the {beta}-hairpin loop 1 of the DENR/Tma22p SUI1 domain as a critical determinant for ribosome recycling and reinitiation, and raise the question of whether MCTS1/Tma20p can promiscuously operate with both DENR/Tma22p and eIF1/Sui1p - two specialized factors that evolved from a common structural scaffold to govern distinct steps in the translation cycle.

Key elements of eukaryotic antiviral immunity are evolutionarily conserved with prokaryotic anti-phage defense systems. The Overcoming Lysogenization Defect (OLD) anti-phage defense system is well characterized in prokaryotes but has remained unknown in eukaryotes. Here, the old-like genes are identified in certain aquatic eukaryotes, including the SAR supergroup, Filasterea, Chytridiomycota, and some metazoan lineages (Placozoa, Cnidaria, Spiralia, Hemichordata, and Cephalochordata). Within molluscs, old-like genes are present in bivalves but absent in gastropods and cephalopods. Interestingly, the filasterean Capsaspora owczarzaki, an endosymbiont of the gastropod Biomphalaria glabrata, encodes putative secreted OLD-like proteins, suggesting it may provide its host with symbiont-mediated antiviral protection. Genomic and transcriptomic analyses revealed that old-like genes vary in copy and intron number and are expressed in some eukaryotic lineages. OLD-like proteins retain key structural features but exhibit a different topology from that of prokaryotes. Structural predictions reveal homodimeric dsDNA-bound architectures analogous to that of the Bacillus cereus VD045 GajA OLD homodimer. Furthermore, phylogenetic analysis divided OLD-like proteins into three distinct clusters, one of which likely represents an ancient inheritance from the ancestral Promethearchaeota (formerly Asgard archaea) host. Together, these findings highlight an unexpected presence of old-like genes in certain aquatic eukaryotes, offering new insights into their evolutionary history and potential role in antiviral defense.

Chemoradiotherapy (CRT) is the standard-of-care therapy for many solid malignancies, yet predictive biomarkers of treatment response remain limited. We identified a germline single nucleotide polymorphism (SNP) in an intrinsically disordered region of the lysine demethylase KDM3C/JMJD1C (p.S464T) that is associated with CRT outcomes in locally advanced rectal cancers (LARC) and head and neck squamous cell carcinoma (LA-HNSCC). In silico modeling with AlphaFold predicted S464T substitution influenced interaction between phosphorylated KDM3C and RNF8 FHA domain. In cellular models, conversion of S464 to T464 increased sensitivity to DNA-damaging agents. S464T substitution impaired damage-induced MDC1-RAP80 signaling and downstream RAP80-BRCA1 colocalization. SNP carrying cells impaired DNA repair causing genotoxic stress that is associated with increased cGAS-cGAMP innate immune signaling and increased apoptosis. Population analyses with the SNP highlighted an increase incidence of UV-induced skin and other cancers, linking inherited variation in the chromatin regulatory gene KDM3C to genome instability, cancer risk, and therapeutic vulnerability.