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PubMed2002

RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO.

The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2-UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA) -- a DNA-polymerase sliding clamp involved in DNA synthesis and repair -- is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination--which additionally requires MMS2, UBC13 and RAD5--and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.

Cell Cycle↗

PubMed2026

Overlapping RAD18- and DNA-binding interfaces in DNA polymerase η contribute to UV-induced DNA damage tolerance.

DNA polymerase η (Polη) bypasses UV-induced pyrimidine dimers and thereby confers tolerance to UV irradiation. Although the C-terminus of Polη has been reported to interact with ubiquitinated PCNA and RAD18, how Polη engages RAD18 is not fully understood. Here, we show that Polη and RAD18 interact through two distinct modes in human cells: a ubiquitinated-PCNA-dependent mode that requires the Polη C-terminus, and an unexpected PCNA-independent mode mediated by its N-terminal region. We focused our subsequent analyses on this newly identified PCNA-independent mode. Using purified recombinant proteins, we demonstrate direct binding of the N-terminal region of human Polη (PolηΔC) to RAD18 in vitro. Although PolηΔC and RAD18 each bound primer-template DNA, we were unable to detect a ternary PolηΔC-RAD18-DNA complex, and DNA competitively inhibited RAD18 binding to both PolηΔC and full-length Polη. Mutational analyses revealed that the DNA-binding and RAD18-binding domains within Polη overlap. A separation-of-function mutant, PolηΔC(K317A), which retains near-normal DNA-binding and polymerase activities but exhibits reduced RAD18 binding in vitro, displayed a diminished ability to rescue the UV sensitivity of Polη-deficient cells. Notably, the detrimental impact of the K317A persisted in a PCNA-binding-defective background but was attenuated in RAD18-knockout cells. These findings demonstrate that RAD18 binding to the N-terminal domain of Polη contributes to efficient bypass of pyrimidine dimers independently of the Polη-PCNA interaction and provide mechanistic insights into how Polη-RAD18 complexes assemble and dissociate during translesion DNA synthesis.

Journal Article↗

PubMed2026

Mechanistic diversity of clamp loading at small DNA gaps.

DNA sliding clamps, including PCNA (proliferating cell nuclear antigen) and the 9-1-1 (RAD9-RAD1-HUS1 in humans) complex, are ring-shaped protein complexes that encircle DNA and serve as central interaction platforms in DNA replication, repair, and checkpoint signaling. While clamp loading at canonical primer-template junctions by AAA+ (ATPases associated with diverse cellular activities) clamp loaders is well established, how clamps are loaded onto physiologically relevant but geometrically constrained DNA intermediates, such as nicks and single-stranded gaps, has remained unclear. Recent cryo-electron microscopy studies reveal that clamp loaders have evolved distinct strategies to overcome these constraints and to specialize for different genomic contexts. At gapped DNA, the eukaryotic clamp loader RFC (replication factor C) engages both 3'- and 5'-recessed DNA ends and can locally unwind DNA, enabling PCNA loading across a wide range of gap sizes. In contrast, the bacterial DnaX clamp loader lacks a 5'-DNA-binding site and does not unwind DNA, instead loading the β-clamp at small gaps (<6 nt) by sharply bending DNA. The checkpoint clamp loader Rad24-RFC (RAD17-RFC in humans) similarly lacks DNA unwinding activity, restricting loading of 9-1-1 clamp to larger gaps (≥6 nt). In a distinct specialization, Ctf18-RFC interacts with the leading-strand DNA polymerase ε, positioning it as a dedicated loader for leading-strand synthesis, whereas Elg1-RFC (ATAD5-RFC in humans) excludes DNA from its chamber and functions as a PCNA unloader. Together, these mechanisms illustrate how clamp loaders are diversified to accommodate DNA structure and replisome context, ensuring coordinated control of genome replication and maintenance.

9-1-1 clamp↗

PubMed2026

Deconstructing the Alternative Lengthening of Telomeres: Integromics Prioritizes Five Master Hubs Dictating Clinical Survival and Therapeutic Vulnerabilities.

The Alternative Lengthening of Telomeres (ALT) pathway drives replicative immortality in aggressive malignancies, particularly sarcomas and gliomas. Clinical ALT stratification has relied on screening for structural ATRX and DAXX mutations. However, this genotypic approach fails to capture the dynamic macro-reprogramming required to sustain ALT. Here, we established and validated a 28-gene transcriptomic signature that captures the ALT-associated transcriptomic phenotype of the ALT phenotype. Using multivariate Cox proportional hazards models and time-dependent ROC analyses, we demonstrate that this signature is a robust, independent predictor of poor overall survival in Sarcoma (SARC) and Lower Grade Glioma (LGG) cohorts, outperforming the prognostic value of traditional ATRX/DAXX mutational status. Genomic mapping revealed this transcriptional synchrony is structurally facilitated by non-random focal clustering on Chromosome 8. To deconstruct the machinery driving this lethal phenotype, we employed an integromic approach, synthesizing protein-protein and metabolic flux networks. Topological algorithms prioritized five indispensable hubs: TP53, ATM, ATR, PCNA, and UBE2I. Gene-metabolite profiling identified PCNA as a bottleneck funneling extreme deoxyribonucleotide (dNTP) demand to sustain break-induced telomeric recombination. To translate these vulnerabilities into actionable treatments, we mapped these hubs to a precision pharmacological network. We propose a multi-targeted strategy combining FDA-approved PARP inhibitors to exploit ATR-mediated synthetic lethality, alongside antimetabolites to induce nucleotide starvation. This study redefines ALT risk stratification and provides a data-driven framework to target and treat resistant ALT-positive tumors.

Alternative Lengthening of Telomeres↗

PubMed2025

The DNA replication checkpoint limits Okazaki fragment accumulation to protect and restart stalled forks.

Understanding how DNA replication forks stall and restart and how the DNA replication checkpoint prevents irreversible fork collapse in molecular detail are crucial for understanding how cells maintain stable genomes and how they prevent the genetic instability that drives cancer. Here, we describe the reconstitution of fork stalling and restart with purified budding yeast proteins. After nucleotide depletion, leading-strand DNA synthesis quickly stops but CMG helicase continues to unwind, and Okazaki fragments continue to initiate on the lagging strand. Incomplete Okazaki fragments sequester PCNA, RFC, and DNA polymerases δ and ε, which prevents normal DNA synthesis restart and exposes nascent DNA to nuclease attack. The DNA replication checkpoint restrains fork progression, which limits this sequestration, protecting stalled forks from collapse and ensuring restart.

DNA Replication↗

PubMed2025

Enzymatic and Structural Roles of Candida albicans Rev1 in DNA Damage Response and Disseminated Candidiasis.

Translesion DNA synthesis (TLS) is a fundamental biological process that enables DNA replication through various lesions to ensure genome stability and to prevent cell death due to replication fork collapse. Rev1, a member of Y-family DNA polymerase (Pol), functions in concert with a B-family enzyme Polζ in promoting TLS through various lesions. Interestingly, for such a function, the catalytic activity of Rev1 seems to be dispensable in Saccharomyces cerevisiae. Unlike Polζ, which possesses robust DNA polymerase activity, biochemical assays suggest that Rev1 predominantly incorporates a "C" opposite any templating residues, but the biological relevance of this activity of Rev1 remains elusive. Here we characterized Rev1 from Candida albicans, an opportunistic fungal pathogen responsible for maximum casualties due to systemic candidiasis in immunosuppressed individuals. Concerted genetic analyses of several Rev1 mutants in various DNA-damaging conditions suggested that in most lesion bypasses except 4-NQO-induced DNA lesions, the catalytic role of Rev1 is not important. However, simultaneous interactions of BRCT and the C-terminal domain of Rev1 with PCNA and Polζ, respectively, enable Rev1 to be essential during TLS. DNA damage recovery and mutagenesis assays further confirmed the lesion-specific roles of various domains of Rev1. Contrary to ex vivo data, animal studies suggested that CaRev1 is dispensable for systemic candidiasis development. We discuss the possible involvement of other TLS DNA polymerases in DNA damage response while C. albicans replicates and establishes itself in the host.

Candida albicans↗

PubMed2022

Suppression of trinucleotide repeat expansion in spermatogenic cells in Huntington's disease.

Trinucleotide repeats (TNRs) are dispersed throughout the human genome. About 20 loci are related to human diseases, such as Huntington's disease (HD). A larger TNR instability is predominantly observed in the paternal germ cells in some TNR disorders. Suppressing the expansion during spermatogenesis can provide a unique opportunity to end the vicious cycle of genetic anticipation. Here, using an in vitro differentiation method to derive advanced spermatogenic cells, we investigated the efficacy of two therapeutic agents, araC (cytarabine) and aspirin, on stabilizing TNRs in spermatogenic cells. Two WT patient-derived induced pluripotent stem cell (iPSC) lines and two HD hiPSC lines, with 44 Q and 180 Q, were differentiated into spermatogonial stem cell-like cells (SSCLCs). Both HD cell lines showed CAG tract expansion in SSCLC. When treated with araC and aspirin, HD1 showed moderate but not statistically significant stabilization of TNR. In HD2, 10 nM of aspirin and araC showed significant stabilization of TNR. All cell lines showed increased DNA damage response (DDR) gene expression in SSCLCs while more genes were significantly induced in HD SSCLC. In HD1, araC and aspirin treatment showed general suppression of DNA damage response genes. In HD2, only FAN1, OGG1, and PCNA showed significant suppression. When the methylation profile of HD cells was analyzed, FAN1 and OGG1 showed significant hypermethylation after the aspirin and araC treatment in SSCLC compared to the control. This study underscores the utility of our in vitro spermatogenesis model to study and develop therapies for TNR disorders such as HD.

Male↗

PubMed2026

Thyroxine enhances breast cancer cell survival and proliferation via TRβ1-Dependent PI3K/AKT signaling.

Thyroid hormones (TH) influence tumor biology through both genomic and non-genomic mechanisms. Specifically, thyroxine (T4) activates signaling pathways linked to cancer progression through interactions with nuclear receptors, such as TRβ1, and membrane receptors, including integrin αvβ3. Nevertheless, the precise role of T4 in breast cancer cell behavior and its underlying molecular mechanisms remain incompletely understood. The effects of physiological concentrations of T4 (10-9 M) on proliferation, cell viability, apoptotic signaling, and activation of intracellular pathways were evaluated in human mammary cell lines. Tumor cell lines (MCF-7 and MDA-MB-231) and the non-tumor mammary epithelial cell line MCF-10A were treated with T4 alone or in combination with the thyroid hormone receptor antagonist 1-850. Cell proliferation was measured using the MTT assay, and viability was determined by trypan blue exclusion. Protein expression and signaling pathways were analyzed by Western blot, including assessment of apoptotic markers (caspases, PARP, Bax, Bcl-2), PCNA, steroid hormone receptors, and signaling mediators such as PI3K, AKT, and ERK. Immunocytochemistry was used to evaluate TRβ1, integrin αvβ3, and Ki67 expression. T4 treatment increased proliferation and survival in hormone-sensitive tumor cells, accompanied by modulation of apoptosis-related proteins and activation of the PI3K/AKT pathway. The antagonist 1-850 selectively attenuated TRβ1-dependent effects, enabling distinction between genomic and integrin-mediated mechanisms. These effects were observed exclusively in hormone-sensitive tumor cells. These findings support a role for T4 in breast cancer progression and identify TH-related signaling pathways as potential therapeutic targets.

Apoptosis↗

PubMed2026

Ovarian H3K27ac remodeling is associated with impaired follicular development in laying hens with fatty liver hemorrhagic syndrome.

Fatty liver hemorrhagic syndrome (FLHS) is a metabolic disease of laying hens that reduces egg production and is accompanied by reproductive impairment, but the ovarian regulatory mechanisms that connect nutritional stress to follicular dysfunction remain unclear. This study examined whether active chromatin remodeling in the ovary is associated with FLHS induced by a high-energy, low-protein (HELP) diet. Hy-Line Brown hens were assigned to a basal diet or HELP diet, and ovarian tissue was collected for histone H3 lysine 27 acetylation (H3K27ac) chromatin immunoprecipitation sequencing and RNA sequencing. The HELP diet reduced laying performance and the numbers of small yellow and hierarchical follicles, indicating compromised follicular development. Genome-wide H3K27ac profiling identified 2,111 regions with lower acetylation and 1,707 regions with higher acetylation in FLHS ovaries. Genes linked to differential H3K27ac regions were enriched in pathways related to oocyte meiosis, cell cycle control, FoxO signaling, gonadotropin-releasing hormone signaling, and steroid hormone biosynthesis. RNA sequencing identified 341 differentially expressed genes, with a predominance of downregulated genes. Integration of chromatin and transcriptome data highlighted folliculogenesis-related genes, including FGF1, FGF9, and MMP10, that showed reduced H3K27ac enrichment together with decreased expression. Super-enhancer analysis further identified 131 regions with reduced H3K27ac signal in FLHS ovaries, including regions located near PCNA and RAP1A, two genes involved in cellular proliferation and survival signaling. Motif enrichment of differential H3K27ac regions implicated Fos, SF-1/NR5A1, and GATA-4 as candidate transcriptional regulators. These findings indicate that HELP diet-induced FLHS is associated with broad attenuation of active ovarian regulatory elements and reduced expression of genes required for follicle growth, tissue remodeling, and steroidogenic function. The study provides an ovarian epigenomic framework for understanding reproductive decline in FLHS-affected laying hens.

Fatty liver hemorrhagic syndrome↗

PubMed2024

SETD8 inhibition targets cancer cells with increased rates of ribosome biogenesis.

SETD8 is a methyltransferase that is overexpressed in several cancers, which monomethylates H4K20 as well as other non-histone targets such as PCNA or p53. We here report novel SETD8 inhibitors, which were discovered while trying to identify chemicals that prevent 53BP1 foci formation, an event mediated by H4K20 methylation. Consistent with previous reports, SETD8 inhibitors induce p53 expression, although they are equally toxic for p53 proficient or deficient cells. Thermal stability proteomics revealed that the compounds had a particular impact on nucleoli, which was confirmed by fluorescent and electron microscopy. Similarly, Setd8 deletion generated nucleolar stress and impaired ribosome biogenesis, supporting that this was an on-target effect of SETD8 inhibitors. Furthermore, a genome-wide CRISPR screen identified an enrichment of nucleolar factors among those modulating the toxicity of SETD8 inhibitors. Accordingly, the toxicity of SETD8 inhibition correlated with MYC or mTOR activity, key regulators of ribosome biogenesis. Together, our study provides a new class of SETD8 inhibitors and a novel biomarker to identify tumors most likely to respond to this therapy.

Humans↗

PubMed2026

Mitotic microhomology-mediated break-induced replication promotes chromoanasynthesis.

Chromoanasynthesis is a form of complex chromosomal rearrangement (CCR) commonly detected in cancers and congenital disorders, but the mechanism underlying its generation remain elusive. Here we develop a single-molecule long-read DNA sequencing approach to characterise ultra-complex mutational events, consistent with chromoanasynthesis, occurring at shortened telomeres and sub-telomeric DNA double-strand breaks in human cells. Our data reveal that chromoanasynthesis is generated by microhomology-mediated break-induced replication (MM-BIR), occurring specifically in mitosis. Surprisingly, this mitotic pathway involves a collaboration between microhomology-mediated end-joining (MMEJ) and BIR, where MMEJ proteins initiate a Polδ-dependent BIR pathway that is regulated by PIF1, POLD3 and PCNA. This pathway is highly prone to template switching and can generate dramatic amplification of genomic loci in a single event. Our findings help explain the extreme mutagenic nature of chromoanasynthesis and establish mitotic MM-BIR as a key driver of CCRs, with important implications for the origin of cancers and congenital disorders.

Humans↗

PubMed2026

Exploiting DNA damage tolerance for precision oncology.

Unresolved DNA lesions trigger replication stress, forcing cancer cells to hijack DNA damage tolerance (DDT) networks, specifically translesion synthesis (TLS) and template switching, to sustain replication. While DDT prevents lethal fork collapse, error-prone TLS drives mutagenesis, tumor evolution, chemoresistance and radioresistance. Proliferating cell nuclear antigen post-translational modifications dynamically govern pathway selection. Cancer cells exploit this plasticity, creating actionable vulnerabilities such as postreplicative single-stranded DNA gaps. Emerging inhibitors targeting TLS polymerases, upstream regulators such as ubiquitin-specific peptidase 1 (USP1), and critical protein-protein interactions offer unprecedented opportunities for precision oncology. By integrating DDT inhibition with biomarkers such as homologous recombination deficiency and tumor mutational burden, we can drive synthetic lethality, sensitize tumors to genotoxic agents, suppress treatment-induced mutagenesis, and potentially enhance responses to immunotherapy.

DDT↗