Flap Endonuclease Research Articles

Innovative Highly Specific Nucleic Acid Isothermal Detection Assay Based on the Polymerization-Coupled Endonuclease Activity of Prokaryotic DNA Polymerase I.

In this study, we developed an innovative highly specific nucleic acid isothermal detection assay based on prokaryotic DNA polymerase I with exquisitely designed fluorescent probes, achieving high sensitivity and 100% specificity within 30 min. The fluorescent nucleic acid probe was designed and constructed based on the specific flap cleavage endonuclease activity of prokaryotic DNA polymerase I (including the Bst, Bsu, Bsm, and Klenow DNA polymerases). The flap endonuclease activity depends on the length of the flap DNA and polymerization activity, which greatly reduces the false-positive rate caused by primer dimerization. This robust assay was also validated by the detection of rotavirus with great specificity and sensitivity. It could be a great alternative to qPCR in the field of point-of-care detection of pathogens.

FEN1 promotes cancer progression of cholangiocarcinoma by regulating the Wnt/β-catenin signaling pathway

PurposeCholangiocarcinoma (CHOL) comprises a cluster of highly heterogeneous malignant biliary tumors. Flap endonuclease-1 (FEN1) is a member of the Rad2 structure-specific nuclease family. This study aimed to explore the biological functions and mechanisms of FEN1 in CHOL. MethodsFEN1 expression was analyzed in tissues of patients with CHOL and FEN1 mutations. We observe the influence of FEN1 on cellular proliferation, migration, and invasion, as well as on DNA damage repair and glycolysis. Western blotting was performed to determine the regulatory mechanism of FEN1 in CHOL progression. ResultsFEN1 was highly expressed in the cancer tissues of CHOL patients. The high mutation rate of FEN1 in CHOL tissues was mainly due to the amplified repeats. FEN1 promotes the proliferation, migration, and invasion of HUCCT1 and QBC939 cells. In addition, FEN1 induced DNA damage repair and aerobic glycolysis in CHOL cells. FEN1 also promoted xenograft tumor growth in vivo. Moreover, we showed that FEN1 mediated the epithelial–mesenchymal transition (EMT) of CHOL. FEN1-mediated EMT was found to be transduced by the Wnt/β-catenin signaling pathway. ConclusionFEN1 was significantly overexpressed in CHOL tissues, and FEN1 regulates the progression of CHOL through the Wnt/β-catenin signaling pathway.

Maintenance of Flap Endonucleases for Long-Patch Base Excision DNA Repair in Mouse Muscle and Neuronal Cells Differentiated In Vitro.

After cellular differentiation, nuclear DNA is no longer replicated, and many of the associated proteins are downregulated accordingly. These include the structure-specific endonucleases Fen1 and DNA2, which are implicated in repairing mitochondrial DNA (mtDNA). Two more such endonucleases, named MGME1 and ExoG, have been discovered in mitochondria. This category of nuclease is required for so-called "long-patch" (multinucleotide) base excision DNA repair (BER), which is necessary to process certain oxidative lesions, prompting the question of how differentiation affects the availability and use of these enzymes in mitochondria. In this study, we demonstrate that Fen1 and DNA2 are indeed strongly downregulated after differentiation of neuronal precursors (Cath.a-differentiated cells) or mouse myotubes, while the expression levels of MGME1 and ExoG showed minimal changes. The total flap excision activity in mitochondrial extracts of these cells was moderately decreased upon differentiation, with MGME1 as the predominant flap endonuclease and ExoG playing a lesser role. Unexpectedly, both differentiated cell types appeared to accumulate less oxidative or alkylation damage in mtDNA than did their proliferating progenitors. Finally, the overall rate of mtDNA repair was not significantly different between proliferating and differentiated cells. Taken together, these results indicate that neuronal cells maintain mtDNA repair upon differentiation, evidently relying on mitochondria-specific enzymes for long-patch BER.

Human DNA ligases I and IIIα as determinants of accuracy and efficiency of base excision DNA repair

Mammalian Base Excision Repair (BER) DNA ligases I and IIIα (LigI, LigIIIα) are major determinants of DNA repair fidelity, alongside with DNA polymerases. Here we compared activities of human LigI and LigIIIα on specific and nonspecific substrates representing intermediates of distinct BER sub-pathways. The enzymes differently discriminate mismatches in the nicked DNA, depending on their identity and position, but are both more selective against the 3′-end non-complementarity. LigIIIα is less active than LigI in premature ligation of one-nucleotide gapped DNA and more efficiently discriminates misinsertion products of DNA polymerase β-catalyzed gap filling, that reinforces a leading role of LigIIIα in the accuracy of short-patch BER. LigI and LigIIIα reseal the intermediate of long-patch BER containing an incised synthetic AP site (F) with different efficiencies, depending on the DNA sequence context, 3′-end mismatch presence and coupling of the ligation reaction with DNA repair synthesis. Processing of this intermediate in the absence of flap endonuclease 1 generates non-canonical DNAs with bulged F site, which are very inefficiently repaired by AP endonuclease 1 and represent potential mutagenic repair products. The extent of conversion of the 5′-adenylated intermediates of specific and nonspecific substrates is revealed to depend on the DNA sequence context; a higher sensitivity of LigI to the sequence is in line with the enzyme structural feature of DNA binding. LigIIIα exceeds LigI in generation of potential abortive ligation products, justifying importance of XRCC1-mediated coordination of LigIIIα and aprataxin activities for the efficient DNA repair.

Dual signal amplification-integrated single-molecule biosensing of flap endonuclease 1 in breast cancer tissues

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that may act as a valuable biomarker for cancer diagnosis. Herein, we construct a dual signal amplification-integrated single-molecule fluorescent biosensor for ultrasensitive detection of FEN1 in breast cancer tissues. We design a triplex DNA containing a flap structure for both recognizing and amplifying FEN1 signal. When FEN1 is present, flap is specifically cleaved by FEN1 and released from triplex DNA to form a short ssDNA strand with free 3' OH terminus, and the resultant ssDNA can serve as the primer and binds to circular template in triplex DNA to trigger rolling circle amplification (RCA), generating a long ssDNA product with abundant repeated activator sequences. The hybridization of the resultant activators with crRNAs subsequently activates Cas12a to catalyze trans-cleavage of Cy5- and BHQ-labeled probes to restore Cy5 fluorescence that can be quantified by single-molecule detection. Notably, only a single triplex DNA is required for both initiating and implementing RCA, greatly simplifying the reaction system and eliminating nonspecific amplification. This biosensor exhibits ultrahigh sensitivity with a detection limit of 2.24 × 10-5 U/μL, facilitating accurate detection of endogenous FEN1 activity with single-cell sensitivity and discrimination of different FEN1 levels in clinical breast cancer tissues.

Colorimetric and photothermal dual readout biosensor for flap endonuclease 1 based on target-prevented gold nanoparticles aggregation

A colorimetric and photothermal dual readout biosensor for Flap endonuclease 1 (FEN1) quantification was developed on the basis of target-prevented gold nanoparticles (AuNPs) aggregation. The exposed 5′-flap of double-flap DNA substrate modified on SAMBs was firstly cleaved by FEN1. Large amount of cleaved 5′-flap remained in the supernatant after simple magnetic separation, which can adsorb on the surface of AuNPs and effectively prevent the dispersed AuNPs from aggregation under high ionic concentration, accompanied with the color changing of the system, which can be recognized by nake eyes easily. The absorption intensity at 528 nm shows a good linear relationship with the increasing FEN1 concentration from 5.0 × 10-3 to 3.1 × 10-2 U μL-1 with a LOD of 1.6 × 10-3 U μL-1 (S/N = 3). Given the aggregated AuNPs have higher photothermal effect than that of the dispersed AuNPs, the target-prevented AuNPs aggregation avoids a sharp increase of temperature for the system under the laser radiation. The temperature change is linearly correlated with the FEN1 concentration in the range of 3.1 × 10-3–6.1 × 10-2 U μL-1 with a LOD of 1.1 × 10-3 U μL-1. The whole detection process can be completed within 1 h. The proposed system had been applied to detect FEN1 concentration in serum samples with satisfied results, which can be applied in resource-limited area easily and quickly.

GENETIC SCREENING REVEALS DEPENDENCY ON FEN1 TO RESIST TEMOZOLOMIDE IN TREATMENT-RESISTANT GLIOBLASTOMA STEM CELLS

Abstract BTFC travel award recipient Glioblastoma (GBM) is a lethal brain tumor. Despite intensive standard of care (SoC) consisting of surgical resection followed by radiation therapy (RT) and chemotherapy with temozolomide (TMZ), tumor relapse is inevitable with a median overall survival of 14.6 months. Therapeutic failure is attributable to the presence of GBM stem-like cells (GSCs), which are highly resistant to genotoxic therapies, self-renew and differentiate to perpetuate intra-tumoral heterogeneity, and seed tumor recurrence. However, no clinical advances have been made in overcoming resistance to therapy in GSCs, and the mechanisms underlying this resistance remain largely unknown. With the advent of CRISPR-Cas9 technology, functional genetic screens have been used to identify genes that regulate survival and sensitivity to TMZ in GSCs. However, they have not been applied to SoC and a comprehensive set of modulators governing resistance to combination treatment has yet to be determined. Here, we perform genome-wide CRISPR-Cas9 screening in patient-derived treatment-sensitive and treatment-resistant GSC models under normal growth conditions or while exposed to in vitro chemoradiotherapy. By integrating genetic dependencies and treatment-based conditional genetic interactions modulating sensitivity to SoC, we identify flap endonuclease 1 (FEN1) as a driver of GSC survival and therapy resistance in treatment-resistant GBM. Functionally, FEN1 inhibition demonstrates specific killing and synergy with TMZ in treatment-resistant GSCs while sparing healthy neural stem cells (NSCs) and treatment-sensitive GSCs. At the molecular level, FEN1 inhibition induces DNA damage and repair specifically in treatment-resistant GBM, posing FEN1 as an attractive therapeutic target in this population of GBM patients which SoC currently fails.

Ultrasensitive turn-on detection of biomarker FEN1 using a CRISPR/Cas13a system integrated with a cleavage-ligation-activation cascade

Flap endonuclease I (FEN1) is a structure-specific nuclease that participates in DNA repair, and its overexpression is related to an increased risk of cancer. Currently, few FEN1 assays can meet the requirements for diagnostic purposes due to their high complexity, low specificity, and limited sensitivity. Herein, we developed a biosensor adopting synthetic biology strategy, called CLAIM (cleavage-ligation-activation-involved monitoring) for FEN1 quantification. CLAIMintegrates ligation-based T7 promoter repair, RNA generation, and CRISPR/Cas13a. FEN1 cleavages the DNA probe with a 5′-flap to generate a nick site that can be ligated to form a T7 promoter. Subsequent transcription produces abundant RNA that can activate Cas13a, generating a fluorescence signal due to the trans-cleavage of Cas13a to a fluorogenic reporter. CLAIM was able to detect FEN1 with low femtomolar sensitivity, and its accuracy was validated using samples of cancer and non-tumorigenic cell lines. Interestingly, we found that the utilization of thermostable T4 DNA ligase is critical to make the cascade of FEN1 cleavage, ligation, and transcription take place in one-pot. CLAIM was also repurposed for validating FEN1 inhibitors, indicating its potential in drug screening. This work provides a diagnostic tool for FEN1 analysis and opens up opportunities for FEN1 biosensing and related drug discovery.

Alleviation of Photoreceptor Degeneration Based on Fullerenols in rd1 Mice by Reversing Mitochondrial Dysfunction via Modulation of Mitochondrial DNA Transcription and Leakage.

Poor therapeutic outcomes of antioxidants in ophthalmologic clinical applications, including glutathione during photoreceptor degeneration in retinitis pigmentosa (RP), are caused by limited anti-oxidative capacity. In this study, fullerenols are synthesized and proven to be highly efficient in vitro radical scavengers. Fullerenol-based intravitreal injections significantly improve the flash electroretinogram and light/dark transition tests performed for 28 days on rd1 mice, reduce the thinning of retinal outer nuclear layers, and preserve the Rhodopsin, Gnat-1, and Arrestin expressions of photoreceptors. RNA-sequencing, RT-qPCR, and Western blotting validate that mitochondrial DNA (mt-DNA)-encoded genes of the electron transport chain (ETC), such as mt-Nd4l, mt-Co1, mt-Cytb, and mt-Atp6, are drastically downregulated in the retinas of rd1 mice, whereas nuclear DNA (n-DNA)-encoded genes, such as Ndufa1 and Atp5g3, are abnormally upregulated. Fullerenols thoroughly reverse the abnormal mt-DNA and n-DNA expression patterns of the ETC and restore mitochondrial function in degenerating photoreceptors. Additionally, fullerenols simultaneously repress Flap endonuclease 1 (FEN1)-mediated mt-DNA cleavage and mt-DNA leakage via voltage-dependent anion channel (VDAC) pores by downregulating the transcription of Fen1 and Vdac1, thereby inactivating the downstream pro-inflammatory cGAS-STING pathway. These findings demonstrate that fullerenols can effectively alleviate photoreceptor degeneration in rd1 mice and serve as a viable treatment for RP.

Post-translational Modifications by Acyl Groups Regulate FEN1's Activities and Play Essential Roles in Cell Proliferation and DNA Repair.

Flap endonuclease 1 (FEN1), well known for its structural-specific nuclease, possessing 5'-flap endonuclease and 5'-3' exonuclease ac-tivities, is mainly involved in DNA replication and repair. Protein lysine acetylation is an important posttranslational modification that could regulate numerous proteins' activity, subcellular localization, protein-protein interac-tion etc., and influences many biological processes. Our previous studies on integrated succinylome profiles found that succinylation and acetylation lev-els of FEN1 would change under different conditions. Succinylation at FEN1 Lys200 site results in the accumulation of damaged DNA and in-creased susceptibility to fork-stalling agents. The interplay with other forms of modification could affects its protein interaction affinity and thus con-tribute to genome stability. This article studied the biological role of FEN1 by acyl modification in HeLa cells. In order to explore the function of FEN1 acylation in cells, we mimicked the presence or ab-sence of acetylation or succinylation by mutating key amino acids to glutam-ic acid and glutamine. We carried out a series of experiments including cell cycle, MTS, enzyme kinetics measurements, immunofluorescence and so on. The absence of acylation of FEN1 leads to the blocked cell cycle process and the reduced efficiency of FEN1 on its DNA substrates, affect-ing the interaction of FEN1 with both repair and replication related proteins and thus its role in the repair of DNA damage. We have veri-fied acyl groups could modify Lys125, Lys252 and Lys254 of FEN1. Acyl-ation level of these three is important for enzyme activity, cell proliferation and DNA damage response, thus contributing to genome stability.

Identification of VPS34-PI(3)P-FEN1-mediated DNA repair pathway as a potential drug target to overcome chemoresistance

Intrinsic or acquired chemoresistance represents a major obstacle in cancer treatment. Multiple mechanisms can contribute to cancer cells' resistance to chemotherapy. Among them, an aberrantly strengthened DNA repair mechanism is responsible for a large proportion of drug resistance to alkylating agents and radiation therapy. In cancer cells, damping overactivated DNA repair system can overcome survival advantages conferred by chromosomal translocations or mutations and lead to cytostatic effects or cytotoxic. Therefore, selectively targeting DNA repair system in cancer cells holds promise for overcoming chemoresistance.In this study, we revealed that the endonuclease Flap Endonuclease 1 (FEN1), essential for DNA replication and repair, directly interacts with phosphatidylinositol 3-phosphate [PI(3)P], and FEN1-R378 is the primary PI(3)P-binding site. PI(3)P-binding deficient FEN1 mutant (FEN1-R378A) cells exhibited abnormal chromosomal structures and were hypersensitized to DNA damage. The PI(3)P-mediated FEN1 functionality was essential for repairing DNA damages caused by multiple mechanisms. Furthermore, VPS34, the major PI(3)P synthesizing enzyme, was negatively associated with patients' survival in various cancer types, and VPS34 inhibitors significantly sensitized chemoresistant cancer cells to genotoxic agents.These findings open up an avenue for counteracting chemoresistance by targeting VPS34-PI(3)P-mediated DNA repair pathway, and call for assessing the efficacy of this strategy in patients suffering from chemoresistance-mediated cancer recurrence in clinical trials.

Androgen receptor knockdown enhances prostate cancer chemosensitivity by down-regulating FEN1 through the ERK/ELK1 signalling pathway.

Flap endonuclease 1 (FEN1) is highly upregulated in prostate cancer and promotes the growth of prostate cancer cells. Androgen receptor (AR) is the most critical determinant of the occurrence, progression, metastasis, and treatment of prostate cancer. However, the effect of FEN1 on docetaxel (DTX) sensitivity and the regulatory mechanisms of AR on FEN1 expression in prostate cancer need to be further studied. Bioinformatics analyses were performed using data from the Cancer Genome Atlas and the Gene Expression Omnibus. Prostate cancer cell lines 22Rv1 and LNCaP were used. FEN1 siRNA, FEN1 overexpression plasmid, and AR siRNA were transfected into cells. Biomarker expression was evaluated by immunohistochemistry and Western blotting. Apoptosis and the cell cycle were explored using flow cytometry analysis. Luciferase reporter assay was performed to verify the target relationship. Xenograft assays were conducted using 22Rv1 cells to evaluate the in vivo conclusions. Overexpression of FEN1 inhibited cell apoptosis and cell cycle arrest in the S phase induced by DTX. AR knockdown enhanced DTX-induced cell apoptosis and cell cycle arrest at the S phase in prostate cancer cells, which was attenuated by FEN1 overexpression. In vivo experiments showed that overexpression of FEN1 significantly increased tumour growth and weakened the inhibitory effect of DTX on prostate tumour growth, while AR knockdown enhance the sensitivity of DTX to prostate tumour. AR knockdown resulted in FEN1, pho-ERK1/2, and pho-ELK1 downregulation, and the luciferase reporter assay confirmed that ELK1 can regulate the transcription of FEN1. Collectively, our studies demonstrate that AR knockdown improves the DTX sensitivity of prostate cancer cells by downregulating FEN1 through the ERK/ELK1 signalling pathway.

FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity

Recombinase polymerase amplification (RPA) running at 37–42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

A DNA Nano Firework for Imaging and Inhibitor Screening of Flap Endonuclease 1 in Living Cells.

In situ observation of changes in the activity of marker proteins in living cells is crucial for both biomarker-based disease diagnosis and drug screening. Flap endonuclease 1 (FEN1) has been recognized as a broad-spectrum cancer biomarker and therapeutic target. However, simple and reliable methods for in situ studying the FEN1 activity changes in living cells are limited. Here, we introduce a nano firework as a fluorescent sensor to sense and report FEN1 activity changes in living cells through FEN1 recognizing the substrates on the surface of the nano firework to release and restore the fluorescence of the prequenched fluorophores. We verified the high selectivity, anti-interference ability, stability, and quantitative performance of the nano firework in tubes and living cells, respectively. A series of controlled experiments have demonstrated that the nano firework could accurately report changes in FEN1 activity in different cells, enabling "sensors in, results out" in the manner of simple addition to the cell culture medium. Using an in silico molecular docking study and experiments, we also explored the ability of the nano firework for rapid screening of FEN1 inhibitors and found two new candidate compounds myricetrin and neoisoliquritin, which could be used as FEN1 inhibitors for further research. These performances of the nano firework suggest that it can be used in high-throughput screening applications, providing a promising tool for biomarker-based new drug discovery.

PCNA Ser46-Leu47 residues are crucial in preserving genomic integrity.

Proliferating cell nuclear antigen (PCNA) is a maestro of DNA replication. PCNA forms a homotrimer and interacts with various proteins, such as DNA polymerases, DNA ligase I (LIG1), and flap endonuclease 1 (FEN1) for faithful DNA replication. Here, we identify the crucial role of Ser46-Leu47 residues of PCNA in maintaining genomic integrity using in vitro, and cell-based assays and structural prediction. The predicted PCNAΔSL47 structure shows the potential distortion of the central loop and reduced hydrophobicity. PCNAΔSL47 shows a defective interaction with PCNAWT leading to defects in homo-trimerization in vitro. PCNAΔSL47 is defective in the FEN1 and LIG1 interaction. PCNA ubiquitination and DNA-RNA hybrid processing are defective in PCNAΔSL47-expressing cells. Accordingly, PCNAΔSL47-expressing cells exhibit an increased number of single-stranded DNA gaps and higher levels of γH2AX, and sensitivity to DNA-damaging agents, highlighting the importance of PCNA Ser46-Leu47 residues in maintaining genomic integrity.

Bacillus subtilis encodes a discrete flap endonuclease that cleaves RNA-DNA hybrids.

The current model for Okazaki fragment maturation in bacteria invokes RNA cleavage by RNase H, followed by strand displacement synthesis and 5' RNA flap removal by DNA polymerase I (Pol I). RNA removal by Pol I is thought to occur through the 5'-3' flap endo/exonuclease (FEN) domain, located in the N-terminus of the protein. In addition to Pol I, many bacteria encode a second, Pol I-independent FEN. The contribution of Pol I and Pol I-independent FENs to DNA replication and genome stability remains unclear. In this work we purified Bacillus subtilis Pol I and FEN, then assayed these proteins on a variety of RNA-DNA hybrid and DNA-only substrates. We found that FEN is far more active than Pol I on nicked double-flap, 5' single flap, and nicked RNA-DNA hybrid substrates. We show that the 5' nuclease activity of B. subtilis Pol I is feeble, even during DNA synthesis when a 5' flapped substrate is formed modeling an Okazaki fragment intermediate. Examination of Pol I and FEN on DNA-only substrates shows that FEN is more active than Pol I on most substrates tested. Further experiments show that ΔpolA phenotypes are completely rescued by expressing the C-terminal polymerase domain while expression of the N-terminal 5' nuclease domain fails to complement ΔpolA. Cells lacking FEN (ΔfenA) show a phenotype in conjunction with an RNase HIII defect, providing genetic evidence for the involvement of FEN in Okazaki fragment processing. With these results, we propose a model where cells remove RNA primers using FEN while upstream Okazaki fragments are extended through synthesis by Pol I. Our model resembles Okazaki fragment processing in eukaryotes, where Pol δ catalyzes strand displacement synthesis followed by 5' flap cleavage using FEN-1. Together our work highlights the conservation of ordered steps for Okazaki fragment processing in cells ranging from bacteria to human.

Construction of dual exponential amplification accompanied by multi-terminal signal output method for convenient detection of tumor biomarker FEN1 activity

As an important 5′-nuclease in DNA replication and damage repair, Flap endonuclease 1 (FEN1) has been considered as a potential tumor biomarker due to its overexpression in different human cancer cells. Here, we developed a convenient fluorescent method based on dual enzymatic repairing exponential amplification accompanied by multi-terminal signal output to realize the rapid and sensitive detection of FEN1. In the presence of FEN1, the double-branched substrate could be cleaved to produce 5′ flap single strand DNA (ssDNA) which subsequently was used as a primer to initiate the dual exponential amplification (EXPAR) to generate abundant ssDNAs (X′ and Y′), then the ssDNAs can respectively hybridize with the 3′ and 5′ ends of the signal probe to form partially complementary double strands (dsDNAs). Subsequently, the signal probe on the dsDNAs could be digested under the assistance of Bst. polymerase and T7 exonuclease, as well as releasing the fluorescence signals. The method displayed high sensitivity with the detection limit of 9.7 × 10−3 U mL−1 (1.94 × 10−4 U) and also exhibited good selectivity towards FEN1 under the challenge from complicated samples including extracts of normal and cancer cells. Furthermore, it was successfully applied to screen FEN1 inhibitors, holding great promise in the screening of potential drugs targeting FEN1. This sensitive, selective and convenient method could be used for FEN1 assay without the complicated nanomaterial synthesis/modification, showing great potential in FEN1- related prediction and diagnosis.

Down-regulation of DNA key protein-FEN1 inhibits OSCC growth by affecting immunosuppressive phenotypes via IFN-γ/JAK/STAT-1

Oral squamous cell carcinoma (OSCC) escape from the immune system is mediated through several immunosuppressive phenotypes that are critical to the initiation and progression of tumors. As a hallmark of cancer, DNA damage repair is closely related to changes in the immunophenotypes of tumor cells. Although flap endonuclease-1 (FEN1), a pivotal DNA-related enzyme is involved in DNA base excision repair to maintain the stability of the cell genome, the correlation between FEN1 and tumor immunity has been unexplored. In the current study, by analyzing the clinicopathological characteristics of FEN1, we demonstrated that FEN1 overexpressed and that an inhibitory immune microenvironment was established in OSCC. In addition, we found that downregulating FEN1 inhibited the growth of OSCC tumors. In vitro studies provided evidence that FEN1 knockdown inhibited the biological behaviors of OSCC and caused DNA damage. Performing multiplex immunohistochemistry (mIHC), we directly observed that the acquisition of critical immunosuppressive phenotypes was correlated with the expression of FEN1. More importantly, FEN1 directly or indirectly regulated two typical immunosuppressive phenotype-related proteins human leukocyte antigen (HLA-DR) and programmed death receptor ligand 1 (PD-L1), through the interferon-gamma (IFN-γ)/janus kinase (JAK)/signal transducer and activator transcription 1 (STAT1) pathway. Our study highlights a new perspective on FEN1 action for the first time, providing theoretical evidence that it may be a potential immunotherapy target for OSCC.

Flap endonuclease Rad27 cleaves the RNA of R-loop structures to suppress telomere recombination.

The long non-coding telomeric RNA transcript TERRA, in the form of an RNA-DNA duplex, regulates telomere recombination. In a screen for nucleases that affects telomere recombination, mutations in DNA2, EXO1, MRE11 and SAE2 cause severe delay in type II survivor formation, indicating that type II telomere recombination is mediated through a mechanism similar to repairing double-strand breaks. On the other hand, mutation in RAD27 results in early formation of type II recombination, suggesting that RAD27 acts as a negative regulator in telomere recombination. RAD27 encodes a flap endonuclease that plays a role in DNA metabolism, including replication, repair and recombination. We demonstrate that Rad27 suppresses the accumulation of the TERRA-associated R-loop and selectively cleaves TERRA of R-loop and double-flapped structures in vitro. Moreover, we show that Rad27 negatively regulates single-stranded C-rich telomeric DNA circles (C-circles) in telomerase-deficient cells, revealing a close correlation between R-loop and C-circles during telomere recombination. These results demonstrate that Rad27 participates in telomere recombination by cleaving TERRA in the context of an R-loop or flapped RNA-DNA duplex, providing mechanistic insight into how Rad27 maintains chromosome stability by restricting the accumulation of the R-loop structure within the genome.

Electrochemical Determination of Flap Endonuclease 1 Activity Amplified by CRISPR/Cas12a Trans‐Cleavage**

AbstractFlap endonuclease 1 (FEN1) participates in the DNA replication and repair processes, which plays an important role in maintaining genome stability. Determination of FEN1 level is highly required for biochemical studies and clinical diagnosis. Herein, a novel electrochemical approach for the detection of FEN1 is developed based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated (Cas) proteins trans‐cleavage. The dumbbell structured DNA substrate, single‐stranded DNA labelled with electrochemical species and three‐dimensional tetrahedral DNA are designed and involved in the reactions. This method displays high sensitivity and good specificity, which can not only monitor FEN1 in various biological samples directly, but also help screen FEN1 inhibitors with potential use for anti‐cancer drug discovery.