DNA Ends Research Articles

Henneguya (Cnidaria: Myxobolidae) species infecting Oligosarcus jenynsii (Characiformes: Characidae) in a Neotropical shallow lake from Argentina: morphological and molecular characterisation.

Two previously undescribed myxozoan species, Henneguya sardellae sp. n. and H. margaritae sp. n., found infecting connective tissues of the Neotropical characid fish Oligosarcus jenynsii (Günther) from Argentina are morphologically and molecularly characterised. Mature spores of H. sardellae sp. n. are ellipsoid, with two, straight and visibly fused caudal appendages cleaved at its blunt terminal end; measuring 33.5 ± 1.2 (30.9-35.5) μm in total length, spore body 17.5 ± 0.6 (16.3-18.6) µm, 7.8 ± 0.4 (7.0-8.8) µm wide and 6.9 ± 0.2 (6.6-7.2) µm thick, with two elongated, unequally-sized polar capsules situated at anterior end, and 11-13 turns of polar tubules. Mature spores of H. margaritae sp. n. are pyriform, with two caudal appendages visible fused together and much longer than spore body, with unequal endings; measuring 35.9 ± 2.8 (29.2-40.7) µm in total length, spore body 11.5 ± 0.9 (9.2-13.0) µm long, 5.8 ± 0.4 (5.1-6.7) µm wide and 5.5 ± 0.2 (5.1-5.8) µm thick, with two polar capsules similar in size, pyriform polar capsules containing polar tubules with 4-5 coils. Both species showed a membraneous sheath surrounding the spore body and caudal appendages; in H. sardellae sp. n. this feature can deploy laterally. Phylogenetic analyses based on SSU rDNA sequences showed that H. sardellae sp. n. and H. margaritae sp. n. clustered with other myxobolids parasitising Characiformes in Brazil, Cichliformes in Mexico and Cyprinodontiformes in Mexico and the United States. The description of these two new species of Henneguya as the first described species of the genus that parasitise freshwater fish in Argentina highlights the importance of further research on the diversity and distribution of myxozoans in this region.

Bisphosphonate's effect on the tongue in adult male albino rats and the possible protective role of rutin: light and scanning electron microscopic study.

Alendronate sodium (ALS) is a nitrogen-containing bisphosphonate used for the treatment of different bone disorders. However, its adverse effect on oral soft tissue has been detected. Rutin (RUT) is natural flavonoid with antioxidant and anti-inflammatory properties. This work aimed to investigate the possible effect of ALS on the tongue of adult male albino rats and to evaluate the possible protective role of RUT. Forty adult male albino rats were equally divided into four groups: group I (control), group II (RUT): Received RUT 50 mg/kg, group III (ALS): Received ALS 1 mg/kg, group IV (ALS+RUT): Received ALS and RUT with the same doses as pervious groups. The drugs were given once daily for 5 weeks. Tongue specimens were taken and processed for light and scanning electron microscopic inspection. ALS treated group revealed structural changes in the tongue in the form of decrease in the height of the filiform papillae with blunt ends, marked atrophy in some papillae with areas of focal loss, loss of some epithelial cells, pyknotic nuclei and cytoplasmic vacuoles in some epithelial cells. The lamina propria showed inflammatory cellular infiltration with congested blood vessels. Statistically, there were highly significant decrease in the number of proliferating cell nuclear antigen immunopositive cells, area percentage of Bcl-2 immunoexpression and highly significant increase in the collagen content compared to control group. Administration of RUT with ALS minimizes these changes. RUT protected the rat tongue against the histological and immunohistochemical changes induced by ALS through its antioxidant and anti-inflammatory properties.

Structural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining

Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ.

NUDT16 regulates CtIP PARylation to dictate homologous recombination repair.

CtIP initiates DNA end resection and mediates homologous recombination (HR) repair. However, the underlying mechanisms of CtIP regulation and how the control of its regulation affects DNA repair remain incompletely characterized. In this study, NUDT16 loss decreases CtIP protein levels and impairs CtIP recruitment to double-strand breaks (DSBs). Furthermore, overexpression of a catalytically inactive NUDT16 mutant is unable to rescue decreased CtIP protein and impaired CtIP recruitment to DSBs. In addition, we identified a novel posttranslational modification of CtIP by ADP-ribosylation that is targeted by a PAR-binding E3 ubiquitin ligase, RNF146, leading to CtIP ubiquitination and degradation. These data suggest that the hydrolase activity of NUDT16 plays a major role in controlling CtIP protein levels. Notably, ADP-ribosylation of CtIP is required for its interaction with NUDT16, its localization at DSBs, and for HR repair. Interestingly, NUDT16 can also be ADP-ribosylated. The ADP-ribosylated NUDT16 is critical for CtIP protein stability, CtIP recruitment to DSBs, and HR repair in response to DNA damage. In summary, we demonstrate that NUDT16 and its PARylation regulate CtIP stability and CtIP recruitment to DSBs, providing new insights into our understanding of the regulation of CtIP-mediated DNA end resection in the HR repair pathway.

Ccq1 restrains Mre11-mediated degradation to distinguish short telomeres from double-strand breaks.

Telomeres protect chromosome ends and are distinguished from DNA double-strand breaks (DSBs) by means of a specialized chromatin composed of DNA repeats bound by a multiprotein complex called shelterin. We investigated the role of telomere-associated proteins in establishing end-protection by studying viable mutants lacking these proteins. Mutants were studied using a Schizosaccharomyces pombe model system that induces cutting of a 'proto-telomere' bearing telomere repeats to rapidly form a new stable chromosomal end, in contrast to the rapid degradation of a control DSB. Cells lacking the telomere-associated proteins Taz1, Rap1, Poz1 or Rif1 formed a chromosome end that was stable. Surprisingly, cells lacking Ccq1, or impaired for recruiting Ccq1 to the telomere, converted the cleaved proto-telomere to a rapidly degraded DSB. Ccq1 recruits telomerase, establishes heterochromatin and affects DNA damage checkpoint activation; however, these functions were separable from protection of the new telomere by Ccq1. In cells lacking Ccq1, telomere degradation was greatly reduced by eliminating the nuclease activity of Mre11 (part of the Mre11-Rad50-Nbs1/Xrs2 DSB processing complex), and higher amounts of nuclease-deficient Mre11 associated with the new telomere. These results demonstrate a novel function for S. pombe Ccq1 to effect end-protection by restraining Mre11-dependent degradation of the DNA end.

PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends

DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.

Regulation of DNA end resection by the Werner syndrome helicase

Regulation of DNA end resection by the Werner syndrome helicase

The role of RNF138 in DNA end resection is regulated by ubiquitylation and CDK phosphorylation

Double-strand breaks (DSBs) are DNA lesions that pose a significant threat to genomic stability. The repair of DSBs by the homologous recombination (HR) pathway is preceded by DNA end resection, the 5' to 3' nucleolytic degradation of DNA away from the DSB. We and others previously identified a role for RNF138, a really interesting new gene finger E3 ubiquitin ligase, in stimulating DNA end resection and HR. Yet, little is known about how RNF138's function is regulated in the context of DSB repair. Here, we show that RNF138 is phosphorylated at residue T27 by cyclin-dependent kinase (CDK) activity during the S and G2 phases of the cell cycle. We also observe that RNF138 is ubiquitylated constitutively, with ubiquitylation occurring in part on residue K158 and rising during the S/G2 phases. Interestingly, RNF138 ubiquitylation decreases upon genotoxic stress. By mutating RNF138 at residues T27, K158, and the previously identified S124 ataxia telangiectasia mutated phosphorylation site (Han etal., 2016, ref. 22), we find that post-translational modifications at all three positions mediate DSB repair. Cells expressing the T27A, K158R, and S124A variants of RNF138 are impaired in DNA end resection, HR activity, and are more sensitive to ionizing radiation compared to those expressing wildtype RNF138. Our findings shed more light on how RNF138 activity is controlled by the cell during HR.

MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner

DNA topoisomerase II (TOP2) is an enzyme that performs a critical function in manipulating DNA topology during replication, transcription, and chromosomal compaction by forming a vital intermediate known as the TOP2-DNA cleavage complex (TOP2cc). Although the TOP2cc is often transient, stabilization can be achieved by TOP2 poisons, a family of anti-cancer chemotherapeutic agents targeting TOP2, such as etoposide (VP-16), and then induce double-strand breaks (DSBs) in cellular DNA. TOP2cc first needs to be proteolyzed before it can be processed by TDP2 for the removal of these protein adducts and to produce clean DNA ends necessary for proper repair. However, the mechanism by which TOP2βcc is proteolyzed has not been thoroughly studied. In this study, we report that after exposure to VP-16, MDM2, a RING-type E3 ubiquitin ligase, attaches to TOP2β and initiates polyubiquitination and proteasomal degradation. Mechanistically, during exposure to VP-16, TOP2β binds to DNA to form TOP2βcc, which promotes MDM2 binding and subsequent TOP2β ubiquitination and degradation, and results in a decrease in TOP2βcc levels. Biologically, MDM2 inactivation abrogates TOP2β degradation, stabilizes TOP2βcc, and subsequently increases the number of TOP2β-concealed DSBs, resulting in the rapid death of cancer cells via the apoptotic process. Furthermore, we demonstrate the combination activity of VP-16 and RG7112, an MDM2 inhibitor, in the xenograft tumor model and in situ lung cancer mouse model. Taken together, the results of our research reveal an underlying mechanism by which MDM2 promotes cancer cell survival in the presence of TOP2 poisons by activating proteolysis of TOP2βcc in a p53-independent manner, and provides a rationale for the combination of MDM2 inhibitors with TOP2 poisons for cancer therapy.

Integration of Cell-Free DNA End Motifs and Fragment Lengths Can Identify Active Genes in Liquid Biopsies.

Multiple studies have shown that cell-free DNA (cfDNA) from cancer patients differ in both fragment length and fragment end motif (FEM) from healthy individuals, yet there is a lack of understanding of how the two factors combined are associated with cancer and gene transcription. In this study, we conducted cfDNA fragmentomics evaluations using plasma from lung cancer patients (n = 12) and healthy individuals (n = 7). A personal gene expression profile was established from plasma using H3K36me3 cell-free chromatin immunoprecipitation sequencing (cfChIP-seq). The genes with the highest expression displayed an enrichment of short cfDNA fragments (median = 19.99%, IQR: 16.94-27.13%, p < 0.0001) compared to the genes with low expression. Furthermore, distinct GC-rich FEMs were enriched after cfChIP. Combining the frequency of short cfDNA fragments with the presence of distinct FEMs resulted in an even further enrichment of the most expressed genes (median = 37.85%, IQR: 30.10-39.49%, p < 0.0001). An in vitro size selection of <150 bp cfDNA could isolate cfDNA representing active genes and the size-selection enrichment correlated with the cfChIP-seq enrichment (Spearman r range: 0.499-0.882, p < 0.0001). This study expands the knowledge regarding cfDNA fragmentomics and sheds new light on how gene activity is associated with both cfDNA fragment lengths and distinct FEMs.

Abstract IA015: Using systems approaches to interrogate DNA double strand break signaling and repair for optimal tumor cell killing

Abstract Repair of DNA double strand breaks (DSBs) occurs through multiple distinct repair pathways including canonical and microhomology-mediated/Pol Q-dependent end joining (c-NHEJ and alt-NHEJ), homologous recombination (HR), and single strand annealing (SSA). Exactly how a particular DSB dictates which pathway is selected for repair, and the extent to which different DSB repair pathways cross-talk with each other is incompletely understood, but appears to be influenced by the specific type of DNA ends, the extent of DNA resection, the cell state and stage of the cell cycle, and the chromatin context around the break. To simultaneously interrogate multiple DNA DSB repair pathways in a systems-based manner at the single cell level, we built upon the pioneering DSB repair reporter systems created by the Jasin, Stark, Scharenberg and Certo labs to create several new CRISPR-based DNA DSB reporter systems that readout the repair of a single genomic DNA DSB by error-free c-NHEJ, mutagenic c- and alt-NHEJ, HR or SSA using multi-color fluorescence-based imaging and flow cytometry approaches. Using these DSB reporter systems we show that competition between short-range and long-range end resection influences the choice between repair by HR versus SSA, demonstrate accepted and novel functions for a variety of DNA damage sensors and repair proteins in NHEJ and HR, and illustrate the feasibility of using this system to interrogate the effects of patient-specific mutations in DNA repair proteins on DNA DSB repair pathway choice. Finally, we show that DNA DSBs created by topoisomerase inhibitor treatment in live but DNA-damaged tumor cells are capable of activating anti-tumor immune responses by signaling through both canonical and non-canonical DNA break sensing and response pathways. Citation Format: Alex Kruswick, Bert van de Kooij, Tiffany R. Emmons, Ganapathy Sriram, Emma D. Martin, Anjali Arvind, Haico van Attikum, Michael B. Yaffe. Using systems approaches to interrogate DNA double strand break signaling and repair for optimal tumor cell killing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA015.

Abstract B010: DNA-PKcs-dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks

Abstract RECQL4, one of the five RecQ helicases in mammalian cells, guards the genome by promoting DNA replication, DNA repair, and telomere maintenance. Mutations in human RECQL4 are associated with three genetic disorders, Rothmund-Thomson syndrome (RTS), Baller-Gerold syndrome (BGS), and RAPADILINO syndrome, which display accelerated aging, developmental abnormalities, and a predisposition to cancer. It is believed that accumulation of DNA damage contributes to the clinical features observed in patients with RECQL4 mutations. Previously, RECQL4 was implicated in promoting the DNA double strand break repair pathway non-homologous end joining (NHEJ), but its role in the pathway remains unresolved. In this study, we report that RECQL4 stabilizes the NHEJ machinery at DSBs to promote repair. Specifically, we find that RECQL4 interacts with the NHEJ core factor DNA-PKcs and the interaction is increased following IR. RECQL4 promotes DNA end bridging mediated by DNA-PKcs and Ku70/80 in vitro and the accumulation/retention of NHEJ factors at DSBs in vivo. Moreover, interaction between DNA-PKcs and the other core NHEJ proteins following IR treatment is attenuated in the absence of RECQL4. These data indicate that RECQL4 promotes the stabilization of the NHEJ factors at DSBs to support formation of the NHEJ long-range synaptic complex. In addition, we observed that the kinase activity of DNA-PKcs is required for accumulation of RECQL4 to DSBs and that DNA-PKcs phosphorylates RECQL4 at six serine/threonine residues. Blocking phosphorylation at these sites reduced the recruitment of RECQL4 to DSBs, attenuated the interaction between RECQL4 and NHEJ factors, destabilized interactions between the NHEJ machinery, and resulted in decreased NHEJ. Collectively, these data illustrate reciprocal regulation between RECQL4 and DNA-PKcs in NHEJ and the potential that dysfunctional NHEJ may play a role in RECQL4-associated genetic disorders and tumorigenesis. Citation Format: Anthony J. Davis. DNA-PKcs-dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B010.

Abstract B033: Direct and sensitive in-situ detection of DNA breaks by STRIDE and its potential as a new biomarker

Abstract DNA damage is usually detected using indirect methods which require active signaling and triggered DNA repair mechanisms. These methods, although relatively sensitive, are prone to false (positive or negative) results. Methods for direct DNA damage detection, on the other hand, suffer from low sensitivity or require DNA isolation, what can result in misleading artefactual readouts of the analysis. Here we present a method called STRIDE (SensiTive Recognition of Individual DNA Ends), which allows for specific, very sensitive in-situ detection of DNA breaks. This fluorescence-based method, in its two variants (dSTRIDE and sSTRIDE), allows selective labelling of double-or single-strand breaks, respectively. STRIDE enables highly sensitive and specific detection and quantification of single and double-strand DNA breaks in cultured cells and FFPE human tissues, both PBMCs as well as solid tumors. STRIDE is superior to classic methods used to measure DNA damage, as it combines a direct detection that is independent of DNA repair mechanisms with an unprecedented sensitivity. With its versatility it can be used in any type of biological material, including liquid biopsies as well as different types of tumors. STRIDE has the potential to become a very useful biomarker reporting about the efficiency of therapies based on DNA damage induction, such as DNA repair inhibitors or radiotherapy. Citation Format: Kamil Solarczyk, Brian Patterson. Direct and sensitive in-situ detection of DNA breaks by STRIDE and its potential as a new biomarker [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B033.

Establishment of the morphological features of stab injuries to the ribs caused by a knife with a blade spine thickness of 0.1 cm

BACKGROUND: The identification of all possible parameters and features of the tool that caused the injuries is of high significance for resolving specific forensic questions in the study of stab injuries to the ribs. However, data on the influence of blade spine thickness on the morphological features of stab injuries to the ribs are lacking.&#x0D; AIM: To determine the morphological features of stab injuries caused by a knife with a blade spine thickness of 0.1 cm at various angles of impact relative to the lamellar structure of the rib.&#x0D; MATERIALS AND METHODS: Experimental works were performed. Injuries were inflicted perpendicularly to the plane of the rib, to a depth of 5.4 cm, with a knife having a blade spine thickness at the penetration level of 0.1 cm. The study analyzed the anterior sections of ribs IV–VI of individuals of both sexes, predominantly male, aged 18–59 years, which is based on an experimental literature review of cutting and stubbing injuries. In total, 60 experiments were conducted, with 20 injuries each inflicted longitudinally, obliquely, and transversely on the rib lamellar structure. The results of the macroscopic observations were recorded and considered for subsequent interpretation.&#x0D; RESULTS: In the macroscopic examination of injuries inflicted at various angles to the lines of the compact layer’s lamellar structure, linear-slit-shaped injuries were registered on the external compact plate. When oriented longitudinally in relation to the long axis of the bone, arcuate slit-shaped injuries were also observed. When a split crack was formed in the blade spine zone, a crack formed from one of the corners of the blunt end of the injury, extending longitudinally and obliquely along the bone’s lamellar structure, with some deviations toward the edge of the bone nearest to the injury. In transverse alignment of the injury to the long axis of the bone, the split crack morphologically corresponded to a fracture in bending deformation, gravitating toward the rib edge. The area influenced by the blade spine showed no macroscopic signs of abrasion of the compact or spongy bone. The blunt end of the injury had a round shape. On the internal compact plate, the shape of the injuries more frequently appeared slit-like, and in some cases serrated, due to the bulging of compact bone areas.&#x0D; CONCLUSION: The study identified significant specific morphological features of stab injuries to the ribs caused by a knife with a blade spine thickness of 0.1 cm, taking into account the lamellar structure of the bone tissue, which allows us to draw conclusions about the knife blade spine thickness.

Kaposi's sarcoma herpesvirus exploits the DNA damage response to circularize its genome.

To establish lifelong, latent infection, herpesviruses circularize their linear, double-stranded, DNA genomes through an unknown mechanism. Kaposi's sarcoma (KS) herpesvirus (KSHV), a gamma herpesvirus, is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV persists in latently infected cells as a multi-copy, extrachromosomal episome. Here, we show the KSHV genome rapidly circularizes following infection, and viral protein expression is unnecessary for this process. The DNA damage response (DDR) kinases, ATM and DNA-PKcs, each exert roles, and absence of both severely compromises circularization and latency. These deficiencies were rescued by expression of ATM and DNA-PKcs, but not catalytically inactive mutants. In contrast, γH2AX did not function in KSHV circularization. The linear viral genomic ends resemble a DNA double strand break, and non-homologous DNA end joining (NHEJ) and homologous recombination (HR) reporters indicate both NHEJ and HR contribute to KSHV circularization. Last, we show, similar to KSHV, ATM and DNA-PKcs have roles in circularization of the alpha herpesvirus, herpes simplex virus-1 (HSV-1), while γH2AX does not. Therefore, the DDR mediates KSHV and HSV-1 circularization. This strategy may serve as a general herpesvirus mechanism to initiate latency, and its disruption may provide new opportunities for prevention of herpesvirus disease.

Impact of Divalent Cations on In-Layer Positional Order of DNA-Based Liquid Crystals: Implications for DNA Condensation.

The layered liquid crystalline phases formed by DNA molecules, which include rigid and flexible segments ("gapped DNA"), enable the study of both end-to-end stacking and side-to-side (helix-to-helix) lateral interactions, forming a model system to study such interactions at physiologically relevant DNA and ion concentrations. The observed layer structure exhibits long-range interlayer and in-layer positional correlations. In particular, the in-layer order has implications for DNA condensation, as it reflects whether these normally repulsive interactions become attractive under certain ionic conditions. Using synchrotron small-angle X-ray scattering measurements, we investigate the impact of divalent Mg2+ cations (in addition to a constant 150 mM Na+) on the stability of the inter- and in-layer DNA ordering as a function of temperature between 5 and 65 °C. DNA constructs with different terminal base pairings were created to mediate the strength of the attractive end-to-end stacking interactions between the blunt ends of the gapped DNA constructs. We demonstrate that the stabilities at a fixed DNA concentration of both interlayer and in-layer order are significantly enhanced even at a few mM Mg2+ concentration. The stabilities are even higher at 30 mM Mg2+; however, a marked decrease is observed at 100 mM Mg2+, suggesting a change in the nature of side-by-side interactions within this Mg2+ concentration range. We discuss the implications of these results in terms of counterion-mediated DNA-DNA attraction and DNA condensation.

Hairpin self-assembly powered by exonuclease III for highly sensitive and cross-validated miRNA-155 detection.

Cancer is one of the most important causes of human death and poses a serious threat to human health. As a cancer biomarker, microRNA-155 (miRNA-155) is highly expressed in various types of cancer tissues and is involved in the proliferation of tumor cells. Therefore, developing a miRNA-155 detection technology with high specificity and sensitivity is of great significance for the early detection, accurate treatment and prognostic evaluation of tumors. Here, we developed a fluorescence detection method using exonuclease III-assisted target cycling and catalytic hairpin assembly (CHA) as a signal amplification technique. This study developed a biosensor for the detection of miRNA-155, utilizing a DNA hairpin (Hp) for target recognition and generating double-stranded DNA (dual-Hp-T). The 3' flat end of the double-stranded DNA can be cleaved by exonuclease III to achieve the target cycle, and a large amount of single-stranded DNA (fuel) can trigger CHA to achieve signal amplification. Simultaneously, the fluorescence resonance energy transfer (FRET) of signal probes with different fluorescence labels on H1 and H2 ends occurs with the CHA reaction. The two fluorescence signals obtained can be used to cross-validate the experimental results. The biosensor exhibits excellent performance of high recovery, high sensitivity and high operability, which can achieve the specific detection of miRNA-155 with a detection limit as low as 8.3 pM. Additionally, the detection efficacy in a human serum environment is also highly satisfactory. This technology provides strong technical support for the development of nucleic acid probes and the diagnosis and treatment of cancer, demonstrating significant practical application value.

Measuring protein stoichiometry with single-molecule imaging in Xenopus egg extracts.

In human cells, DNA double-strand breaks are rapidly bound by the highly abundant non-homologous end joining (NHEJ) factor Ku70/Ku80 (Ku). Cellular imaging and structural data revealed a single Ku molecule is bound to a free DNA end and yet the mechanism regulating Ku remains unclear. Here, we describe how to utilize the cell-free Xenopus laevis egg extract system in conjunction with single-molecule microscopy to investigate regulation of Ku stoichiometry during non-homologous end joining. Egg extract is an excellent model system to study DNA repair as it contains the soluble proteome including core and accessory NHEJ factors, and efficiently repairs double-strand breaks in an NHEJ-dependent manner. To examine the Ku stoichiometry in the extract system, we developed a single-molecule photobleaching assay, which reports on the number of stable associated Ku molecules by monitoring the intensity of fluorescently labeled Ku molecules bound to double-stranded DNA over time. Photobleaching is distinguishable as step decreases in fluorescence intensity and the number of photobleaching events indicate fluorophore stoichiometry. In this paper we describe sample preparation, experimental methodology, and data analysis to discern Ku stoichiometry and the regulatory mechanism controlling its loading. These approaches can be readily adopted to determine stoichiometry of molecular factors within other macromolecular complexes.

Olaparib combined with CDK12-IN-3 to promote genomic instability and cell death in ovarian cancer.

Large-scale phase III clinical trials of Olaparib have revealed benefits for ovarian cancer patients with BRCA gene mutations or homologous recombination deficiency (HRD). However, fewer than 50% of ovarian cancer patients have both BRCA mutations and HRD. Therefore, improving the effect of Olaparib in HR-proficient patients is of great clinical value. Here, a combination strategy comprising Olaparib and CDK12-IN-3 effectively inhibited the growth of HR-proficient ovarian cancer in cell line, patient-derived organoid (PDO), and mouse xenograft models. Furthermore, the combination strategy induced severe DNA double-strand break (DSB) formation, increased NHEJ activity in the G2 phase, and reduced HR activity in cancer cells. Mechanistically, the combination treatment impaired Ku80 poly(ADP-ribosyl)ation (PARylation) and phosphorylation, resulting in PARP1-Ku80 complex dissociation. After dissociation, Ku80 occupancy at DSBs and the resulting Ku80-primed NHEJ activity were increased. Owing to Ku80-mediated DNA end protection, MRE11 and Rad51 foci formation was inhibited after the combination treatment, suggesting that this treatment suppressed HR activity. Intriguingly, the combination strategy expedited cGAS nuclear relocalization, further suppressing HR and, conversely, increasing genomic instability. Moreover, the inhibitory effect on cell survival persisted after drug withdrawal. These findings provide a rationale for the clinical application of CDK12-IN-3 in combination with Olaparib.

Xrs2/NBS1 promote end-bridging activity of the MRE11-RAD50 complex

DNA double strand breaks (DSBs) can be detrimental to the cell and need to be efficiently repaired. A first step in DSB repair is to bring the free ends in close proximity to enable ligation by non-homologous end-joining (NHEJ), while the more precise, but less available, repair by homologous recombination (HR) requires close proximity of a sister chromatid. The human MRE11-RAD50-NBS1 (MRN) complex, Mre11-Rad50-Xrs2 (MRX) in yeast, is involved in both repair pathways. Here we use nanofluidic channels to study, on the single DNA molecule level, how MRN, MRX and their constituents interact with long DNA and promote DNA bridging. Nanofluidics is a suitable method to study reactions on DNA ends since no anchoring of the DNA end(s) is required. We demonstrate that NBS1 and Xrs2 play important, but differing, roles in the DNA tethering by MRN and MRX. NBS1 promotes DNA bridging by MRN consistent with tethering of a repair template. MRX shows a “synapsis-like” DNA end-bridging, stimulated by the Xrs2 subunit. Our results highlight the different ways MRN and MRX bridge DNA, and the results are in agreement with their key roles in HR and NHEJ, respectively, and contribute to the understanding of the roles of NBS1 and Xrs2 in DSB repair.