Genomic Instability Research Articles

Tolerance of Oncogene-Induced Replication Stress: A Fuel for Genomic Instability.

Activation of oncogenes disturbs a wide variety of cellular processes and induces physiological dysregulation of DNA replication, widely referred to as replication stress (RS). Oncogene-induced RS can cause replication forks to stall or collapse, thereby leading to DNA damage. While the DNA damage response (DDR) can provoke an anti-tumor barrier to prevent the development of cancer, a small subset of cells triggers replication stress tolerance (RST), allowing precancerous cells to survive, thereby promoting clonal expansion and genomic instability (GIN). Genomic instability (GIN) is a hallmark of cancer, driving genetic alterations ranging from nucleotide changes to aneuploidy. These alterations increase the probability of oncogenic events and create a heterogeneous cell population with an enhanced ability to evolve. This review explores how major oncogenes such as RAS, cyclin E, and MYC induce RS through diverse mechanisms. Additionally, we delve into the strategies employed by normal and cancer cells to tolerate RS and promote GIN. Understanding the intricate relationship between oncogene activation, RS, and GIN is crucial to better understand how cancer cells emerge and to develop potential cancer therapies that target these vulnerabilities.

DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death.

Deleterious germline DDX41 variants constitute the most common inherited predisposition disorder linked to myeloid neoplasms (MNs). The role of DDX41 in hematopoiesis and how its germline and somatic mutations contribute to MNs remain unclear. Here we show that DDX41 is essential for erythropoiesis but dispensable for the development of other hematopoietic lineages. Using stage-specific Cre models for erythropoiesis, we reveal that Ddx41 knockout in early erythropoiesis is embryonically lethal, while knockout in late-stage terminal erythropoiesis allows mice to survive with normal blood counts. DDX41 deficiency induces a significant upregulation of G-quadruplexes (G4), noncanonical DNA structures that tend to accumulate in the early stages of erythroid precursors. We show that DDX41 co-localizes with G4 on the erythroid genome. DDX41 directly binds to and dissolves G4, which is significantly compromised in MN-associated DDX41 mutants. Accumulation of G4 by DDX41 deficiency induces erythroid genome instability, defects in ribosomal biogenesis, and upregulation of p53. However, p53 deficiency does not rescue the embryonic death of Ddx41 hematopoietic-specific knockout mice. In parallel, genome instability also activates the cGas-Sting pathway, which is detrimental to survival since cGas-deficient and hematopoietic-specific Ddx41 knockout mice are viable without detectable hematologic phenotypes, although these mice continue to show erythroid ribosomal defects and upregulation of p53. These findings are further supported by data from a DDX41 mutated MN patient and human iPSC-derived bone marrow organoids. Our study establishes DDX41 as a G4 dissolver, essential for erythroid genome stability and suppressing the cGAS-STING pathway.

Structure-based discovery of first inhibitors targeting the helicase activity of human PIF1.

PIF1 is a conserved helicase and G4 DNA binding and unwinding enzyme, with roles in genome stability. Human PIF1 (hPIF1) is poorly understood, but its functions can become critical for tumour cell survival during oncogene-driven replication stress. Here we report the discovery, via an X-ray crystallographic fragment screen (XChem), of hPIF1 DNA binding and unwinding inhibitors. A structure was obtained with a 4-phenylthiazol-2-amine fragment bound in a pocket between helicase domains 2A and 2B, with additional contacts to Valine 258 from domain 1A. The compound makes specific interactions, notably through Leucine 548 and Alanine 551, that constrain conformational adjustments between domains 2A and 2B, previously linked to ATP hydrolysis and DNA unwinding. We next synthesized a range of related compounds and characterized their effects on hPIF1 DNA-binding and helicase activity in vitro, expanding the structure activity relationship (SAR) around the initial hit. A systematic analysis of clinical cancer databases is also presented here, supporting the notion that hPIF1 upregulation may represent a specific cancer cell vulnerability. The research demonstrates that hPIF1 is a tractable target through 4-phenylthiazol-2-amine derivatives as inhibitors of its helicase action, setting a foundation for creation of a novel class of anti-cancer therapeutics.

Fungal L-Methionine Biosynthesis Pathway Enzymes and Their Applications in Various Scientific and Commercial Fields.

L-methionine (L-Met) is one of the nine proteinogenic amino acids essential for humans since, in human cells, there are no complete pathways for its biosynthesis from simple precursors. L-Met plays a crucial role in cellular function as it is required for proper protein synthesis, acting as an initiator. Additionally, this amino acid participates in various metabolic processes and serves as a precursor for the synthesis of S-adenosylmethionine (AdoMet), which is involved in the methylation of DNA molecules and phospholipids, as well as in maintaining genome stability. Due to its importance, fungal L-methionine biosynthesis pathway enzymes are being intensively studied. This review presents the current state of the art in terms of their cellular function, usefulness as molecular markers, antifungal targets, or industrial approaches.

OTUD5 promotes end-joining of deprotected telomeres by promoting ATM-dependent phosphorylation of KAP1S824

Appropriate repair of damaged DNA and the suppression of DNA damage responses at telomeres are essential to preserve genome stability. DNA damage response (DDR) signaling consists of cascades of kinase-driven phosphorylation events, fine-tuned by proteolytic and regulatory ubiquitination. It is not fully understood how crosstalk between these two major classes of post-translational modifications impact DNA repair at deprotected telomeres. Hence, we performed a functional genetic screen to search for ubiquitin system factors that promote KAP1S824 phosphorylation, a robust DDR marker at deprotected telomeres. We identified that the OTU family deubiquitinase (DUB) OTUD5 promotes KAP1S824 phosphorylation by facilitating ATM activation, through stabilization of the ubiquitin ligase UBR5 that is required for DNA damage-induced ATM activity. Loss of OTUD5 impairs KAP1S824 phosphorylation, which suppresses end-joining mediated DNA repair at deprotected telomeres and at DNA breaks in heterochromatin. Moreover, we identified an unexpected role for the heterochromatin factor KAP1 in suppressing DNA repair at telomeres. Altogether our work reveals an important role for OTUD5 and KAP1 in relaying DDR-dependent kinase signaling to the control of DNA repair at telomeres and heterochromatin.

Mycobacterium smegmatis NucS-promoted DNA mismatch repair involves limited resection by a 5'-3' exonuclease and is independent of homologous recombination and NHEJ.

The MutSL mismatch repair (MMR) systems in bacteria and eukaryotes correct mismatches made at the replication fork to maintain genome stability. A novel MMR system is represented by the EndoMS/NucS endonuclease from Actinobacterium Corynebacterium glutamicum, which recognizes mismatched substrates in vitro and creates dsDNA breaks at the mismatch. In this report, a genetic analysis shows that an M. smegmatis ΔnucS strain could be complemented by expression of wild type NucS protein, but not by one deleted of its last five amino acids, a region predicted to be critical for binding to the β-clamp at the replication fork. Oligo-recombineering was then leveraged to deliver defined mismatches to a defective hygromycin resistance gene on the M. smegmatis chromosome. We find that NucS recognizes and repairs G-G, G-T, and T-T mismatches in vivo, consistent with the recognition of these same mismatches in C. glutamicum in vitro, as well as mutation accumulation studies done in M. smegmatis. Finally, an assay that employs an oligo that promotes the generation of two mismatches in close proximity on the chromosome shows that a NucS-promoted cut is processed by a 5'-3' exonuclease (or 5'-Flap endonuclease)and that NucS-promoted MMR is independent of both homologous recombination and non-homologous end-joining.

Double-strand breaks in facultative heterochromatin require specific movements and chromatin changes for efficient repair

DNA double-strand breaks (DSBs) must be properly repaired within diverse chromatin domains to maintain genome stability. Whereas euchromatin has an open structure and is associated with transcription, facultative heterochromatin is essential to silence developmental genes and forms compact nuclear condensates, called polycomb bodies. Whether the specific chromatin properties of facultative heterochromatin require distinct DSB repair mechanisms remains unknown. Here, we integrate single DSB systems in euchromatin and facultative heterochromatin in Drosophila melanogaster and find that heterochromatic DSBs rapidly move outside polycomb bodies. These DSB movements coincide with a break-proximal reduction in the canonical heterochromatin mark histone H3 Lysine 27 trimethylation (H3K27me3). We demonstrate that DSB movement and loss of H3K27me3 at heterochromatic DSBs depend on the histone demethylase dUtx. Moreover, loss of dUtx specifically disrupts completion of homologous recombination at heterochromatic DSBs. We conclude that DSBs in facultative heterochromatin require dUtx-mediated loss of H3K27me3 to promote DSB movement and repair.

Characterization of Two Novel PNKP Splice-Site Variants in a Proband With Microcephaly, Intellectual Disability, and Multiple Malformations.

Polynucleotide kinase phosphatase (PNKP), encoded by the PNKP gene, is a DNA processing enzyme involved in double-strand break and single-strand break repair pathways, which are essential for genome stability and for the correct development and maintenance of human nervous system. PNKP biallelic loss-of-function variants have been associated with a broad spectrum of neurological anomalies, ranging from congenital microcephaly with intellectual disability and seizures (MCSZ), to later onset forms of ataxia-oculomotor apraxia (AOA4) or peripheral neuropathy (CMT2B2). We report the atypical clinical manifestations of a patient with severe microcephaly, short stature, developmental delay, conductive hearing loss, and tracheoesophageal malformation, in the absence of seizures. Whole exome sequencing analysis identified two novel, compound heterozygous splice-site variants in the PNKP gene (NM_007254.4): c.1448+1G > A and c.199-8_199-5del. To demonstrate the effect of both variants on the splicing process and prove their pathogenicity, we performed a hybrid minigene assay, which successfully highlighted a deleterious impact on the transcript, particularly regarding the c.199-8_199-5del variant. The uncommon clinical features of the proband and the identification of two newly associated pathogenic variants add further evidence to the allelic and phenotypic heterogeneity of the PNKP locus.

Critical roles of Dpb3-Dpb4 sub-complex of DNA polymerase epsilon in DNA replication, genome stability, and pathogenesis of Candida albicans.

This study explored the role of DNA polymerase epsilon, especially its non-essential structural subunits in Candida albicans biology. Apart from their role in DNA replication and genome stability, the Dpb3-Dpb4 subcomplex regulates morphological switching and virulence. Since the defective strain is locked in filamentous form and is avirulent, the complex may be targeted for anti-fungal drug development.

Effect of CAT gene polymorphism on sensitivity of human lymphocytes to genotoxic effect of organochlorine pesticide in vitro

Introduction. Over recent decades, toxicogenetic studies have focused on the issues of genome instability under the action of genotoxicants, taking into account biomarkers of sensitivity. The question about the genotoxic potential of chlorpyrifos remains open, since both positive and negative effects have been revealed in various tests. The aim of the study is the investigation of sensitivity of donor peripheral blood lymphocytes to chlorpyrifos in vitro and evaluation of the contribution of polymorphism of antioxidant defense system genes (CAT (rs1001179), SOD2 (rs4880)) to the response of human cells to the action of genotoxicant. Materials and methods. The DNA damaging effect of chlorpyrifos was assessed on lymphocytes from fifty two donors using DNA-comet assay with metabolic activation (+S9) and without it (–S9). The study of cytotoxic effects of chlorpyrifos on human lymphocytes was carried out using an automatic fluorescent cell analyzer ADAMII LS. Results. Chlorpyrifos had a pronounced cytotoxic effect on lymphocytes in most donors in the absence of metabolic activation system. With increasing concentration of the pesticide in the medium and time of cultivation, the viability of lymphocytes decreased, and the proportion of cells in late apoptosis and necrosis increased. Positive genotoxic effects were found on the cells of 33 donors (-S9). In the presence of the S9, mild but statistically significant effects were detected only on cells from 2 donors. % DNA values in the comet tail after exposure to the pesticide varied for cells from different donors. In the absence of metabolic activation, a statistically significant increase in the level of DNA damage was found in cells of individuals with genotype AA (homozygote for the minor allele) for the CAT G262A catalase gene (rs1001179), compared with homozygote for the dominant GG allele. Limitations. The genotoxicity of chlorpyryfos was studied in vitro. Conclusion. The results of the study shown cytotoxic and genotoxic effects of chlorpyrifos. The sensitivity of lymphocytes from different donors to the pesticide was found to be significantly different. The association of the level of DNA damage under exposure of chlorpyrifos in vitro with the G262A polymorphism of the catalase gene was found. The research also confirms the possibility of using a model test-system with peripheral blood lymphocytes to assess the potential genetic risk for humans and to study the contribution of gene polymorphism to individual sensitivity to the action of genotoxicants.

Genomic instability and genetic heterogeneity in aging: insights from clonal hematopoiesis (CHIP), monoclonal gammopathy (MGUS), and monoclonal B-cell lymphocytosis (MBL).

Aging is a multifaceted process characterized by a gradual decline in physiological function and increased susceptibility to a range of chronic diseases. Among the molecular and cellular mechanisms driving aging, genomic instability is a fundamental hallmark, contributing to increased mutation load and genetic heterogeneity within cellular populations. This review explores the role of genomic instability and genetic heterogeneity in aging in the hematopoietic system, with a particular focus on clonal hematopoiesis of indeterminate potential (CHIP), monoclonal gammopathy of undetermined significance (MGUS), and monoclonal B-cell lymphocytosis (MBL) as biomarkers. CHIP involves the clonal expansion of hematopoietic stem cells with somatic mutations. In contrast, MGUS is characterized by the presence of clonal plasma cells producing monoclonal immunoglobulins, while MBL is characterized by clonal proliferation of B cells. These conditions are prevalent in the aging population and serve as measurable indicators of underlying genomic instability. Studying these entities offers valuable insights into the mechanisms by which somatic mutations accumulate and drive clonal evolution in the hematopoietic system, providing a deeper understanding of how aging impacts cellular and tissue homeostasis. In summary, the hematopoietic system serves as a powerful model for investigating the interplay between genomic instability and aging. Incorporating age-related hematological conditions into aging research, alongside other biomarkers such as epigenetic clocks, can enhance the precision and predictive power of biological age assessments. These biomarkers provide a comprehensive view of the aging process, facilitating the early detection of age-related diseases and hopefully enabling personalized healthcare strategies.

Dysfunctional copper homeostasis in Caenorhabditis elegans affects genomic and neuronal stability

While copper (Cu) is an essential trace element for biological systems due to its redox properties, excess levels may lead to adverse effects partly due to overproduction of reactive species. Thus, a tightly regulated Cu homeostasis is crucial for health. Cu dyshomeostasis and elevated labile Cu levels are associated with oxidative stress and neurodegenerative disorders, but the underlying mechanisms have yet to be fully characterized. Here, we used Caenorhabditis elegans loss-of-function mutants of the Cu chaperone ortholog atox-1 and the Cu binding protein ortholog ceruloplasmin to model Cu dyshomeostasis, as they display a shifted ratio of total Cu towards labile Cu. We applied highly selective and sensitive techniques to quantify metabolites associated to oxidative stress with focus on mitochondrial integrity, oxidative DNA damage and neurodegeneration all in the context of a disrupted Cu homeostasis. Our novel data reveal elevated oxidative stress, compromised mitochondria displaying reduced ATP levels and cardiolipin content. Cu dyshomeostasis further induced oxidative DNA damage and impaired DNA damage response as well as neurodegeneration characterized by behavior and neurotransmitter analysis. Our study underscores the essentiality of a tightly regulated Cu homeostasis as well as mitochondrial integrity for both genomic and neuronal stability.

RAD51 recombinase and its paralogs: Orchestrating homologous recombination and unforeseen functions in protozoan parasites

The DNA of protozoan parasites is highly susceptible to damage, either induced by environmental agents or spontaneously generated during cellular metabolism through reactive oxygen species (ROS). Certain phases of the cell cycle, such as meiotic recombination, and external factors like ionizing radiation (IR), ultraviolet light (UV), or chemical genotoxic agents further increase this susceptibility. Among the various types of DNA damage, double-stranded breaks (DSBs) are the most critical, as they are challenging to repair and can result in genetic instability or cell death. DSBs caused by environmental stressors are primarily repaired via one of two major pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). In multicellular eukaryotes, NHEJ predominates, but in unicellular eukaryotes such as protozoan parasites, HR seems to be the principal mechanism for DSB repair. The HR pathway is orchestrated by proteins from the RAD52 epistasis group, including RAD51, RAD52, RAD54, RAD55, and the MRN complex. This review focuses on elucidating the diverse roles and significance of RAD51 recombinase and its paralogs in protozoan parasites, such as Acanthamoeba castellanii, Entamoeba histolytica (Amoebozoa), apicomplexan parasites (Chromalveolata), Naegleria fowleri, Giardia spp., Trichomonas vaginalis, and trypanosomatids (Excavata), where they primarily function in HR. Additionally, we analyze the diversity of proteins involved in HR, both upstream and downstream of RAD51, and discuss the implications of these processes in parasitic protozoa.

DYSREGULATION OF M6A RNA METHYLATION, R-LOOPS AND THE DNA DAMAGE RESPONSE IN PAEDIATRIC HIGH-GRADE GLIOMAS

Abstract AIMS Paediatric high-grade gliomas (pHGG) are virtually incurable malignant brain tumours. The regulation of R- loops is necessary for ensuring genomic stability. The formation of R-loops at DNA damage sites promotes DNA repair via m6A methylation of the RNA strand. We aimed to investigate whether m6A methylation, R-loop homeostasis and the DNA damage response is dysregulated in pHGG and whether inhibitors, such as ATM inhibitors, could be potential therapeutics for pHGG. METHOD To assess the global levels of m6A, R-loops, yH2AX, total ATM and phosphorylated ATM in paediatric HGG cell lines compared to control human astrocytes, immunocytochemistry was initially performed. Immunocyto- chemistry was also performed to assess localisation between m6A and R-loops, R-loops and yH2AX, R-loops and total/phosphorylated ATM, m6A and total/phosphorylated ATM, and total/phosphorylated ATM and yH2AX in pHGG cell lines and human astrocytes. We are also exploring two ATM inhibitors, AZD1390 and AZD0156, and are currently investigating their effects on m6A methylation, R-loops and the DNA damage response in pHGG. As a molecular mechanism involving the tumour suppressor ARID1A was recently discovered that is crucial for maintaining genomic stability, we are also exploring the role of this protein in pHGG. We are also investigating the potential involvement of m6A readers in the pathogenesis of pHGG. RESULTS Immunocytochemistry revealed that dysregulation of global m6A, R-loop, yH2AX and ATM occurs in pHGG cell lines as higher levels of expression was generally observed compared to human astrocytes. Moreover, m6A, R-loops, yH2AX and ATM appear to colocalise as high Pearson’s correlation coefficient values were obtained. Experiments exploring ATM inhibitors, ARID1A and m6A readers in pHGG are currently in progress, however the results will be presented. CONCLUSION It appears that dysregulation of m6A methylation, R-loop homeostasis and the DNA damage response occurs in pHGG and that molecular mechanisms involving m6A methylation and R-loops could be impaired.

Double-Edged Sword Effect of Diet and Nutrition on Carcinogenic Molecular Pathways in Breast Cancer.

Environmental exposure to a mixture of chemical xenobiotics acts as a double-edged sword, promoting or suppressing tumorigenesis and the development of breast cancer (BC). Before anything else, we are what we eat. In this review, we highlight both "the good" and "the bad" sides of the daily human diet and dietary patterns that could influence BC risk (BCR) and incidence. Thus, regularly eating new, diversified, colorful, clean, nutrient-rich, energy-boosting, and raw food, increases apoptosis and autophagy, antioxidation, cell cycle arrest, anti-inflammation, and the immune response against BC cells. Moreover, a healthy diet could lead to a reduction in or the inhibition of genomic instability, BC cell stemness, growth, proliferation, invasion, migration, and distant metastasis. We also emphasize that, in addition to beneficial compounds, our food is more and more contaminated by chemicals with harmful effects, which interact with each other and with endogenous proteins and lipids, resulting in synergistic or antagonistic effects. Thus, a healthy and diverse diet, combined with appropriate nutritional behaviors, can exert anti-carcinogenic effects and improve treatment efficacy, BC patient outcomes, and the overall quality of life of BC patients.

Causes and consequences of DNA double-stranded breaks in cardiovascular disease.

The genome, whose stability is essential for survival, is incessantly exposed to internal and external stressors, which introduce an estimated 104 to 105 lesions, such as oxidation, in the nuclear genome of each mammalian cell each day. A delicate homeostatic balance between the generation and repair of DNA lesions maintains genomic stability. To initiate transcription, DNA strands unwind to form a transcription bubble and provide a template for the RNA polymerase II (RNAPII) complex to synthesize nascent RNA. The process generates DNA supercoils and introduces torsional stress. To enable RNAPII processing, the supercoils are released by topoisomerases by introducing strand breaks, including double-stranded breaks (DSBs). Thus, DSBs are intrinsic genomic features of gene expression. The breaks are quickly repaired upon processing of the transcription. DNA lesions and damaged proteins involved in transcription could impede the integrity and efficiency of RNAPII processing. The impediment, which is referred to as transcription stress, not only could lead to the generation of aberrant RNA species but also the accumulation of DSBs. The latter is particularly the case when topoisomerase processing and/or the repair mechanisms are compromised. The DSBs activate the DNA damage response (DDR) pathways to repair the damaged DNA and/or impose cell cycle arrest and cell death. In addition, the release of DSBs into the cytosol activates the cytosolic DNA-sensing proteins (CDSPs), which along with the nuclear DDR pathways induce the expression of senescence-associated secretory phenotype (SASP), cell cycle arrest, senescence, cell death, inflammation, and aging. The primary stimulus in hereditary cardiomyopathies is a mutation(s) in genes encoding the protein constituents of cardiac myocytes; however, the phenotype is the consequence of intertwined complex interactions among numerous stressors and the causal mutation(s). Increased internal DNA stressors, such as oxidation, alkylation, and cross-linking, are expected to be common in pathological conditions, including in hereditary cardiomyopathies. In addition, dysregulation of gene expression also imposes transcriptional stress and collectively with other stressors provokes the generation of DSBs. In addition, the depletion of nicotinamide adenine dinucleotide (NAD), which occurs in pathological conditions, impairs the repair mechanism and further facilitates the accumulation of DSBs. Because DSBs activate the DDR pathways, they are expected to contribute to the pathogenesis of cardiomyopathies. Thus, interventions to reduce the generation of DSBs, enhance their repair, and block the deleterious DDR pathways would be expected to impart salubrious effects not only in pathological states, as in hereditary cardiomyopathies but also aging.

The histone chaperones ASF1 and HIRA are required for telomere length and 45S rDNA copy number homeostasis.

Genome stability is significantly influenced by the precise coordination of chromatin complexes that facilitate the loading and eviction of histones from chromatin during replication, transcription, and DNA repair processes. In this study, we investigate the role of the Arabidopsis H3 histone chaperones ANTI-SILENCING FUNCTION 1 (ASF1) and HISTONE REGULATOR A (HIRA) in the maintenance of telomeres and 45S rDNA loci, genomic sites that are particularly susceptible to changes in the chromatin structure. We find that both ASF1 and HIRA are essential for telomere length regulation, as telomeres are significantly shorter in asf1a1b and hira mutants. However, these shorter telomeres remain localized around the nucleolus and exhibit a comparable relative H3 occupancy to the wild type. In addition to regulating telomere length, ASF1 and HIRA contribute to silencing 45S rRNA genes and affect their copy number. Besides, ASF1 supports global heterochromatin maintenance. Our findings also indicate that ASF1 transiently binds to the TELOMERE REPEAT BINDING 1 protein and the N terminus of telomerase in vivo, suggesting a physical link between the ASF1 histone chaperone and the telomere maintenance machinery.

The epistatic relationship of Drosophila melanogaster CtIP and Rif1 in homology-directed repair of DNA double-strand breaks.

Double-strand breaks (DSBs) are genotoxic DNA lesions that pose significant threats to genomic stability, necessitating precise and efficient repair mechanisms to prevent cell death or mutations. DSBs are repaired through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR), which includes homologous recombination (HR) and single-strand annealing (SSA). CtIP and Rif1 are conserved proteins implicated in DSB repair pathway choice, possibly through redundant roles in promoting DNA end-resection required for HDR. Although the roles of these proteins have been well-established in other organisms, the role of Rif1 and its potential redundancies with CtIP in Drosophila melanogaster remain elusive. To examine the roles of DmCtIP and DmRif1 in DSB repair, this study employed the direct repeat of white (DR-white) assay, tracking across indels by decomposition (TIDE) analysis, and P{wIw_2 kb 3'} assay to track repair outcomes in HR, NHEJ, and SSA, respectively. These experiments were performed in DmCtIPΔ/Δ single mutants, DmRif1Δ/Δ single mutants, and DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants. This work demonstrates significant defects in both HR and SSA repair in DmCtIPΔ/Δ and DmRif1Δ/Δ single mutants. However, experiments in DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants reveal that DmCtIP is epistatic to DmRif1 in promoting HDR. Overall, this study concludes that DmRif1 and DmCtIP do not perform their activities in a redundant pathway, but rather DmCtIP is the main driver in promoting HR and SSA, most likely through its role in end resection.

Aberrant FAM135B attenuates the efficacy of chemotherapy in colorectal cancer by modulating SRSF1-mediated alternative splicing.

Oxaliplatin is the frontline chemotherapy drug for the treatment of colorectal cancer (CRC) and its insensitivity is a major limitation on therapeutic efficacy. Genomic instability is the prominent feature of CRC and is considered to correlate with response to treatments. However, the underlying mechanism of insensitivity to oxaliplatin (L-OHP) remains largely unclear. Herein, sequence similarity 135 family member B (FAM135B) is identified as a frequently mutated gene in CRC and is critical for CRC proliferation and impaired response to L-OHP by controlling SRSF1-mediated alternative splicing. Specifically, FAM135B promotes the nuclear translocation of SRSF1 by synergistically binding with SRPK1 and regulates SRSF1-mediated splicing of DNA repair genes. FAM135B-induced exon IV inclusion of FAAP20 mediates its binding with FACNA and enhances the functional integrity of the FA core complex, thereby activating the FA pathway and resulting in inter-strand crosslink (ICL) lesion repair and L-OHP insensitivity. These findings reveal that the FAM135B-SRSF1 axis-mediated splicing contributes to DNA repair and chemotherapeutic insensitivity in CRC. Targeting FAM135B represents a potential strategy for CRC treatment.

RNA Polymerase II is a Polar Roadblock to a Progressing DNA Fork.

DNA replication and transcription occur simultaneously on the same DNA template, leading to inevitable conflicts between the replisome and RNA polymerase. These conflicts can stall the replication fork and threaten genome stability. Although numerous studies show that head-on conflicts are more detrimental and more prone to promoting R-loop formation than co-directional conflicts, the fundamental cause for the RNA polymerase roadblock polarity remains unclear, and the structure of these R-loops is speculative. In this work, we use a simple model system to address this complex question by examining the Pol II roadblock to a DNA fork advanced via mechanical unzipping to mimic the replisome progression. We found that the Pol II binds more stably to resist removal in the head-on configuration, even with minimal transcript size, demonstrating that the Pol II roadblock has an inherent polarity. However, an elongating Pol II with a long RNA transcript becomes an even more potent and persistent roadblock while retaining the polarity, and the formation of an RNA-DNA hybrid mediates this enhancement. Surprisingly, we discovered that when a Pol II collides with the DNA fork head-on and becomes backtracked, an RNA-DNA hybrid can form on the lagging strand in front of Pol II, creating a topological lock that traps Pol II at the fork. TFIIS facilitates RNA-DNA hybrid removal by severing the connection of Pol II with the hybrid. We further demonstrate that this RNA-DNA hybrid can prime lagging strand replication by T7 DNA polymerase while Pol II is still bound to DNA. Our findings capture basal properties of the interactions of Pol II with a DNA fork, revealing significant implications for transcription-replication conflicts.