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PubMed2026

A versatile cGAMP reporter reveals principles of cGAS activation by DNA damage and chromosome instability.

cGAS is the primary innate immune DNA sensor. On binding DNA, cGAS generates cGAMP, ultimately driving inflammation. Although normally silenced on self-DNA, genotoxic stress can activate cGAS, proposed to be mediated by micronuclei, chromosome bridges and DNA:RNA hybrids. However, mechanistically, this is poorly understood due to a lack of sensitive and selective single-cell cGAS activation assays. Here we solve this with an improved cGAMP reporter for microscopy, flow cytometry and biochemical assays. Strikingly, we find that genotoxic stress-mediated cGAS activation is a rare event that is not driven by enrichment on micronuclei and occurs by mechanisms that vary in dependence on the genotoxic stress. Following chromosome mis-segregation, cGAS activation correlates with bridge association but, notably, ionizing radiation activates cGAS independently of bridges. Whereas simple DNA:RNA hybrids are inert, more complex structures such as R-loops activate cGAS. Our work revises the cGAS signalling framework and introduces a flexible tool to examine it.

Nucleotidyltransferases↗
PubMed2025

Crosstalk between chromatin state and ATM signalling in DNA damage-induced transcription stress.

The DNA Damage Response (DDR) is a highly regulated process that safeguards genomic integrity against DNA lesions. Increasing evidence supports a reciprocal relationship between damaged chromatin architecture and the signalling pathways that coordinate the DDR. However, the mechanisms underlying this interplay in response to transcription-blocking DNA lesions remain largely unexplored. Here, we show that stalling of RNA polymerase II (RNAPII) at such lesions induces local chromatin acetylation, mediated primarily by the histone acetyltransferase p300. The resulting chromatin relaxation stimulates the dissociation of mature co-transcriptional spliceosomes from nascent RNA and promotes RNA:DNA hybrid (R-loop) formation, leading to ATM activation. In turn, activated ATM modulates chromatin conformation by phosphorylating histone H2A.X and triggering p38MAPK/MSK1-dependent histone H3S10 phosphorylation. Our findings highlight the cross-regulation between chromatin state and ATM signalling as a key component of the cellular response to transcription stress.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2025

Lamin A/C loss promotes R-loop-mediated genomic instability and poor survival in small-cell lung cancer.

Lamin A/C (LMNA), a key component of the nuclear envelope, is essential for maintaining nuclear integrity and genome organization [W. Xie et al., Curr. Biol. 26, 2651-2658 (2016)]. While LMNA dysregulation has been implicated in genomic instability across cancer and aging, the underlying mechanisms remain poorly understood [S. Graziano et al., Nucleus 9, 258-275 (2018)]. Here, we define a mechanistic role for LMNA in preserving genome stability in small-cell lung cancer (SCLC), a malignancy marked by extreme genomic instability [N. Takahashi et al., Cancer Res. Commun. 2, 503-517 (2022)]. LMNA depletion promotes R-loop accumulation, transcription-replication conflicts, replication stress, DNA breaks, and micronuclei formation. Mechanistically, LMNA deficiency disrupts nuclear pore complex organization, specifically reducing phenylalanine-glycine (FG)-nucleoporin incorporation, resulting in impaired RNA export and nuclear retention of RNA. LMNA expression is repressed by EZH2 and reexpressed during SCLC differentiation from neuroendocrine (NE) to non-NE states, and low LMNA levels correlate with poor clinical outcomes. These findings establish LMNA as a key regulator of nuclear transport and genome integrity, linking nuclear architecture to SCLC progression and therapeutic vulnerability.

Lamin Type A↗
PubMed1978

The organization of ribosomal RNA genes in the mitochondrial DNA of Tetrahymena pyriformis strain ST.

1. We have constructed a physical map of the mtDNA of Tetrahymena pyriformis strain ST using the restriction endonucleases EcoRI, PstI, SacI, HindIII and HhaI. 2. Hybridization of mitochondrial 21 S and 14 S ribosomal RNA to restriction fragments of strain ST mtDNA shows that this DNA contains two 21-S and only one 14-S ribosomal RNA genes. By S1 nuclease treatment of briefly renatured single-stranded DNA the terminal duplication-inversion previously detected in this DNA (Arnberg et al. (1975) Biochim. Biophys. Acta 383, 359--369) has been isolated and shown to contain both 21-S ribosomal RNA genes. 14 S ribosomal RNA hybridizes to a region in the central part of the DNA, about 8000 nucleotides or 20% of the total DNA length apart from the nearest 21 S ribosomal RNA gene. 3. We have confirmed this position of the three ribosomal RNA genes by electron microscopical analysis of DNA . RNA hybrid molecules and R-loop molecules. 4. Hybridization of 21 S ribosomal RNA with duplex mtDNA digested either with phage lambda-induced exonuclease or exonuclease III of Escherichia coli, shows that the 21-S ribosomal RNA genes are located on the 5'-ends of each DNA strand. Electron microscopy of denaturated mtDNA hybridized with a mixture of 14-S and 21-S ribosomal RNAs show that the 14 S ribosomal RNA gene has the same polarity as the nearest 21 S ribosomal RNA gene. 5. Tetrahymena mtDNA is (after Saccharomyces mtDNA) the second mtDNA in which the two ribosomal RNA cistrons are far apart and the first mtDNA in which one of the ribosomal RNA cistrons is duplicated.

Animals↗
PubMed2025

Determining the biochemical function of type IV CRISPR ribonucleoprotein complexes and accessory proteins.

Type IV CRISPR systems are phylogenetically diverse and poorly understood. However, recently, major strides have been made toward understanding type IV-A systems. In type IV-A systems, a multi-subunit ribonucleoprotein complex, called the Csf complex, uses a CRISPR-derived guide to bind double-stranded DNA, forming an R-loop to which a helicase called CRISPR-associated DinG (CasDinG) is recruited. It is proposed that the ATP-dependent helicase activity of CasDinG then unwinds duplex DNA near the targeting site, impairing RNA transcription, and gene expression. Here we describe methods used to investigate the type IV-A system from Pseudomonas aeruginosa strain 83 including a plasmid clearance assay, expression and purification of type IV ribonucleoprotein complexes and proteins, nucleic acid binding assays, and CasDinG helicase assays. These methods provide a foundation for future work aimed at understanding these enigmatic systems.

Ribonucleoproteins↗
PubMed2026

Pan-cancer single-cell atlas of immunotherapy response identifies ZNF385A as a regulator of immune evasion in small cell lung cancer.

Although immune checkpoint inhibitors (ICIs) have revolutionized the treatment landscape of solid tumors, response rates in patients with small cell lung cancer (SCLC) remain limited, and acquired resistance is highly prevalent. The underlying mechanisms of this immunotherapy resistance remain to be fully elucidated. Clinically, SCLC typically manifests as an "immune-cold" tumor, characterized by a low abundance of CD8+ T cell infiltration and the rare formation of tertiary lymphoid structures (TLS). While DNA damage repair (DDR) is closely linked to innate immune responses, how DDR networks orchestrate the SCLC immune microenvironment remains obscure. In this study, we integrated single-cell transcriptomic data (comprising 344,447 high-quality cells) from six cancer types (BCC, CRC, HCC, HNSCC, iCCA, and SCLC). Our comparative analysis revealed a fundamental depletion of TLS-associated cellular subpopulations (e.g., CXCL13+ CD8+ T cells, HLA-DRB5+ B cells, and CXCL9+ dendritic cells) in SCLC, which was significantly correlated with aberrant DDR activity. Through high-dimensional weighted gene co-expression network analysis (hdWGCNA), we identified ZNF385A as the core hub gene within the DDR-associated module. ZNF385A is highly expressed in SCLC and is associated with poorer prognosis. In vitro, ZNF385A depletion suppressed SCLC cell proliferation and induced apoptosis, accompanied by R-loop accumulation and activation of cGAS-STING signaling, indicating a potential link between ZNF385A, genomic stability and tumor-intrinsic innate immune signaling. Collectively, these findings identify ZNF385A as a potential regulator associated with TLS deficiency and immune evasion in SCLC.

Immunotherapy resistance↗
PubMed2026

Type IV-C CRISPR-Cas effector complexes recognize double-stranded DNA and switch on collateral cleavage of ssDNA and RNA.

Type IV-C CRISPR-Cas systems remain enigmatic compared to other class 1 systems. Here, we expand the type IV-C catalog, identifying two phylogenetically distinct clades primarily found in archaea (IV-C1) or bacteria (IV-C2), distinguishable by the Cas10IVc subunit architecture. We functionally and structurally characterize type IV-C1 systems from Thermococcus onnurineus (Ton) and Pyrococcus abyssi (Pab). Type IV-C complexes assemble with crRNAs derived from distinct CRISPR arrays and recognize a 5'-GGG-3' protospacer adjacent motif (PAM) to bind double-stranded DNA targets. Target recognition activates the HD domain of Cas10IVc, triggering metal-dependent collateral cleavage of single-stranded DNA and RNA. This behavior is explained by allosteric alignment of the HD active site, triggered by PAM-dependent R-loop formation, as revealed by cryo-EM. Together, our findings suggest that type IV-C systems provide immunity via non-specific cleavage of nucleic acids generated during mobile genetic element replication or transcription.

CP: molecular biology↗
PubMed2026

RAD54L coordinates the nucleolar DNA damage response to maintain rDNA stability.

The nucleolus is organized around actively transcribed ribosomal RNA genes (rDNA), where high RNA polymerase I (Pol I) activity creates intrinsic susceptibility to replication stress and DNA damage. Here, we identify the DNA translocase RAD54L as a critical regulator of the nucleolar DNA damage response (nDDR) to rDNA double-strand breaks (DSBs) and replication stress. We show that RAD54L localizes to the nucleolus under basal conditions and is recruited to nucleolar caps following CRISPR-Cas9-induced rDNA-DSBs to promote repair. RAD54L loss results in persistent RAD51 foci, increased nucleolar γH2AX, and micronuclei formation, indicating defective resolution of rDNA lesions and genome instability. Under baseline conditions and replication stress induced by the Pol I transcription inhibitor CX-5461, RAD54L limits the accumulation of ssDNA and coordinates nDDR signaling. We further show that rDNA-DSBs induce RNA polymerase II-dependent RNA-DNA hybrids (R-loops) at intergenic rDNA regions, which facilitate nucleolar reorganization and cap formation and repair factor recruitment. Together, these findings establish RAD54L as a key regulator that coordinates replication stress response and rDNA repair, maintaining rDNA stability and genome integrity.

DNA, Ribosomal↗
PubMed2026

Distinct functions of mammalian RAD51 paralogs in genome maintenance.

RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are evolutionarily conserved essential proteins for cell survival and genome maintenance. RAD51 paralogs were originally identified to play a role in homologous recombination-mediated repair of DNA double-strand breaks (DSBs). However, investigations over the last decade have uncovered new roles of RAD51 paralogs beyond DSB repair in replication stress responses, including replication fork progression, fork stability, and its restart. Recent structural studies have not only uncovered the molecular architecture of previously known RAD51 paralog complexes but also identified novel paralog complex assemblies, providing mechanistic insights into their various genome-maintenance functions. Additionally, a role for RAD51 paralogs in resolving R-loops has been identified, and studies with cancer-associated variants suggest that RAD51 paralogs are potential determinants of cancer susceptibility and therapeutic responses. In the present review, we highlight the recently deciphered structures and novel functions of RAD51 paralog complexes and discuss the clinical and therapeutic implications.

Rad51 Recombinase↗
PubMed1978

DNA clones containing mouse immunoglobulin kappa chain genes isolated by in vitro packaging into phage lambda coats.

Endonuclease EcoRI-digested DNAs from BALB/c mouse embryos and MOPC 321 (a kappa chain secretor) myeloma were fractionated by agarose gel electrophoresis, and the DNA fragments containing part or all of the MOPC 321 kappa chain structural gene sequences were visualized by the Southern gel blotting technique using as the hybridization probes pCRI plasmids containing all or part of the enzymatically synthesized cDNA transcripts of the MOPC 321 kappa chain mRNA. The clear differences observed in the hybridization patterns of the two DNAs are in agreement with our previously reported results obtained with endonuclease BamHI and confirms that the sequence arrangement of kappa chain genes is different in the embryo and myeloma cells. We have cloned most of the kappa-sequence-positive EcoRI DNA fragments in Charon 4A phage by using the highly efficient in vitro phage lambda DNA packaging method, and we have characterized the cloned mouse DNA sequences by agarose gel blotting and R-loop mapping in electron microscopy. These studies identified, among others, one EcoRI DNA fragment which contains both variable and constant immunoglobulin kappa-gene sequences and is present only in the myeloma DNA. The two sequences are separated by a 2.8-kbase intron. We tentatively conclude that the kappa gene sequences on this DNA fragment underwent somatic rearrangement.

Animals↗
PubMed1979

Electron microscopic analysis of transcription: mapping of initiation sites and direction of transcription.

An electron microscope technique is described that allows rapid characterization of transcription in vitro. DNA is transcribed with Escherichia coli RNA polymerase in vitro, and the RNA is hybridized to its template. Measurement of the resulting transcription R-loop molecules allows accurate mapping of transcription initiation sites (promoter sites) and analysis of the direction and rate of transcription and the level of transcription from each initiation site. The two major early promoters pR and pL of bacteriophage lambda have been mapped within 0.1-0.3 map units of the known positions and three additional sites have been confirmed. Six transcription initiation sites have been preliminarily mapped on plasmid pSF2124 DNA.

Bacteriophage lambda↗
PubMed1979

The rat serum albumin gene: analysis of cloned sequences.

The rat serum albumin gene has been isolated from a recombinant library containing the entire rat genome cloned in the lambda phage Charon 4A. Preliminary R-loop and restriction analysis has revealed that this gene is split into at least 14 fragments (exons) by 13 intervening sequences (introns), and that it occupies a minimum of 14.5 kilobases of genomic DNA.

Animals↗
PubMed1979

Cloning immunoglobulin gamma 2b chain gene of mouse: characterization and partial sequence determination.

DNA from newborn mice was digested with restriction endonuclease EcoRI, and a 6.6-kilobase fragment encoding immunoglobulin gamma 2b chain mRNA derived from MPC 11 myeloma was enriched about 100-fold by RPC-5 column chromatography and agarose gell electrophoresis. The 6.6-kilobase fragment was cloned with lambda gt WES.lambda B as EK2 vector. The cloned phage (lambda WES.IgH22) contained the constant region gene of the gamma 2b chain but not the variable region gene of MPC 11 mRNA. The constant region genes of the other gamma chains (i.e., gamma 1, gamma 2a, and gamma 3) were not present in lambda gt WES.IgH22 DNA. R-loop mapping indicates that the gamma 2b chain structural gene is divided into two parts (330 +/- 60 SD base pairs and 930 +/- 110 SD base pairs) by an intervening sequence (360 +/- 100 SD base pairs). The nucleotide sequence around the junction of the hinge region and CH2 domain was determined and shown to match the amino acid sequence of the initial part of the CH2 domain of the gamma 2b chain. The base sequence upstream from the junction, however, is unrelated to the amino acid sequence of the CH1 domain and the hinge region of all the gamma chains whose sequences have been determined. These results indicate that the gamma 2b chain gene is interrupted at the junction of the hinge region and CH2 domain by an intervening sequence. The existence of two more intervening sequences, one between the CH1 domain and the hinge region and the other between the CH2 and CH3 domains, is discussed.

Animals↗
PubMed2026

G-quadruplex structures as regulators of cellular processes and drivers of genome instability in cancer.

G-quadruplexes (G4s) are essential regulatory structures whose biological functions are inseparable from their potential to destabilize the genome. They play critical roles in transcription, replication and chromatin architecture, yet they also contribute to the genomic instability that fuels cancer. This dual role is an inherent consequence of where G4s form in the genome. G4s are enriched at highly active regulatory regions, including promoters, replication origins and topologically associated domain boundaries, where their controlled formation and resolution by helicases and topoisomerases support normal genome function. When this control is lost, the same features that make G4s functional become harmful, leading to R-loop and G-loop accumulation, replication fork stalling and increased conflicts between transcription and replication. Topoisomerase activity, which normally relieves supercoiling stress, can instead generate the DNA double-strand breaks that characterize cancer genomes. Persistent G4 structures also promote micronuclei formation and cytoplasmic DNA accumulation, activating the cGAS-STING innate immune signaling pathway. Here, we discuss these mechanisms and present pan-cancer genomic analyses showing that these processes operate broadly across human tumors. Therapeutically targeting G4s requires balancing their essential regulatory roles with their pathological effects. Understanding this tension is therefore essential for exploiting G4s as therapeutic targets across cancer types.

DEAH-box helicases↗
PubMed1979

Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173.

The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images

Animals↗
PubMed2025

Sensitive, direct detection of non-coding off-target base editor unwinding and editing in primary cells.

Base editors create precise nucleotide changes in DNA, but their off-target activity remains challenging to quantify. Here, we develop and deploy a direct, in cellulo sequencing assay that simultaneously measures both Cas9-mediated unwinding and deaminase editing of genomic DNA (beCasKAS). Our strategy nominates >460-fold more potential off-target sites than other methods by enriching for Cas9-dependent R-loops immediately preceding editing. Using beCasKAS in primary human T-cells, we observe that mRNA-encoded ABE8e and PAMless ABE8e-SpRY base editors have distinct off-target profiles that can be mitigated by optimizing mRNA dose. Finally, we combine beCasKAS with base-resolution deep learning models to risk-stratify off-target edits by their likelihood of epigenetic dysregulation. Collectively, beCasKAS offers a sensitive and facile tool to optimize the balance between base editor on- and off-target activity.

Journal Article↗
PubMed2026

Advances in CRISPR Base Editing: From Molecular Evolution to Therapeutic Applications in Genomic Medicine.

CRISPR-Cas9 systems revolutionized gene editing, but inherent drawbacks, namely DNA double-strand breaks (DSBs) and the difficulty of achieving precise repairs (due to low HDR efficiency), led researchers to invent new, more accurate gene editing tools. Base editing represents a significant leap forward, enabling targeted single-nucleotide conversions directly on the DNA without DSBs or donor templates. The core technology involves fusing catalytically dead or nickase Cas proteins to DNA deaminase enzymes. Cytosine base editors (CBEs) convert C•G to T•A pairs, while adenine base editors (ABEs) change A•T to G•C. These editors exploit the deaminase function within the R-loop structure formed by Cas binding and co-opt endogenous DNA repair mechanisms for precision. While offering improved efficiency and editing precision, base editing faces persistent challenges, such as off-target effects, bystander edits, delivery and ethical concerns. Continuous engineering efforts have refined these tools, enhancing accuracy, expanding targetability and reducing unwanted edits. The base editing arsenal has also broadened to include C-to-G base editors (CGBEs), dual A&C editors and versions targeting organelles. Successful preclinical studies demonstrating the correction of mutations responsible for the disease have paved the way for clinical trials, which are now testing therapies for conditions like sickle cell disease, β-thalassaemia and hypercholesterolemia using various delivery systems. This review explores CRISPR base editing's origins, mechanisms of action, potential therapies and current restrictions, pointing to its broadening impact on medical genetics.

Humans↗