Mechanistic insights into FEN1-mediated drug sensitivity and risk signature in colon cancer: An integrative bioinformatics study.

The overexpression of Flap endonuclease 1 (FEN1) has been implicated in drug resistance and prognosis across various cancer types. However, the precise role of FEN1 in colon cancer remains to be fully elucidated. In this study, we employed comprehensive datasets from The Cancer Genome Atlas, Gene Expression Omnibus, and Human Protein Atlas to examine FEN1 expression and assess its correlation with clinical pathology and prognosis in colon cancer. We utilized the pRRophetic algorithm to evaluate drug sensitivity and performed differential expression analysis to identify genes associated with FEN1-mediated drug sensitivity. Gene set enrichment analysis was conducted to further investigate these genes. Additionally, single-cell sequencing analysis was employed to explore the relationship between FEN1 expression and functional states. Cox regression analysis was implemented to construct a prognostic model, and a nomogram for prognosis was developed. Our analysis of The Cancer Genome Atlas and Gene Expression Omnibus datasets revealed a significant upregulation of FEN1 in colon cancer. However, while FEN1 expression showed no notable correlation with prognosis, it displayed associations with metastasis. Single-cell sequencing analysis further confirmed a positive correlation between FEN1 expression and colon cancer metastasis. Furthermore, we detected marked discrepancies in drug responsiveness between the High_FEN1 and Low_FEN1 groups, identifying 342 differentially expressed genes. Enrichment analysis showed significant suppression in processes related to DNA replication, spliceosome, and cell cycle pathways in the Low_FEN1 group, while the calcium signaling pathway, cAMP signaling pathway, and other pathways were activated. Of the 197 genes differentially expressed and strongly linked to FEN1 expression, 39 were significantly implicated in colon cancer prognosis. Finally, we constructed a risk signature consisting of 5 genes, which, when combined with drug treatment and pathological staging, significantly improved the prediction of colon cancer prognosis. This study offers novel insights into the interplay among FEN1 expression levels, colon cancer metastatic potential, and sensitivity to therapeutic agents. Furthermore, we successfully developed a multi-gene prognostic risk signature derived from FEN1.

Target mediated bioreaction to engineer surface vacancy effect on Bi2O2S nanosheets for photoelectrochemical detection of FEN1.

Photoelectrochemistry represents a promising technique for bioanalysis, though its application for the detection of Flap endonuclease 1 (FEN1) has not been tapped. Herein, this work reports the exploration of creating oxygen vacancies (Ov) in situ onto the surface of Bi2O2S nanosheets via the attachment of dopamine (DA), which underlies a new anodic PEC sensing strategy for FEN1 detection in label-free, immobilization-free and high-throughput modes. In connection to the target-mediated rolling circle amplification (RCA) reaction for modulating the release of the DA aptamer to capture DA, the detection system showed good performance toward FEN1 analysis with a linear detection range of 0.001-10 U/mL and a detection limit of 1.4 × 10-4 U/mL (S/N = 3). This work features the bioreaction engineered surface vacancy effect of Bi2O2S nanosheets as a PEC sensing strategy, which allows a simple, easy to perform, sensitive and selective method for the detection of FEN1. This sensing strategy might have wide applications in versatile bioasssays, considering the diversity of a variety of biological reactions may produce the DA aptamer.

PARG-deficient tumor cells have an increased dependence on EXO1/FEN1-mediated DNA repair.

Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.

FEN1 Inhibition as a Potential Novel Targeted Therapy against Breast Cancer and the Prognostic Relevance of FEN1.

To improve breast cancer treatment and to enable new strategies for therapeutic resistance, therapeutic targets are constantly being studied. Potential targets are proteins of DNA repair and replication and genomic integrity, such as Flap Endonuclease 1 (FEN1). This study investigated the effects of FEN1 inhibitor FEN1-IN-4 in combination with ionizing radiation on cell death, clonogenic survival, the cell cycle, senescence, doubling time, DNA double-strand breaks and micronuclei in breast cancer cells, breast cells and healthy skin fibroblasts. Furthermore, the variation in the baseline FEN1 level and its influence on treatment prognosis was investigated. The cell lines show specific response patterns in the aspects studied and have heterogeneous baseline FEN1 levels. FEN1-IN-4 has cytotoxic, cytostatic and radiosensitizing effects, expressed through increasing cell death by apoptosis and necrosis, G2M share, senescence, double-strand breaks and a reduced survival fraction. Nevertheless, some cells are less affected by the cytotoxicity and fibroblasts show a rather limited response. In vivo, high FEN1 mRNA expression worsens the prognosis of breast cancer patients. Due to the increased expression in breast cancer tissue, FEN1 could represent a new tumor and prognosis marker and FEN1-IN-4 may serve as a new potent agent in personalized medicine and targeted breast cancer therapy.

Ligase detection reaction amplification-activated CRISPR-Cas12a for single-molecule counting of FEN1 in breast cancer tissues.

We construct a simple fluorescent biosensor for single-molecule counting of flap endonuclease 1 (FEN1) based on ligase detection reaction (LDR) amplification-activated CRISPR-Cas12a. This biosensor exhibits excellent selectivity and high sensitivity with a detection limit (LOD) of 1.31 × 10-8 U. Moreover, it can be employed to screen the FEN1 inhibitors and quantitatively measure the FEN1 activity in human cells and breast cancer tissues, holding great promise in clinical diagnosis and drug discovery.

In Situ Track-Generated DNA Walker for AND-Gate Logic Imaging of Telomerase and Flap Endonuclease 1 Activities in Living Cells.

In situ monitoring of the actions of correlated enzymes in living cells is crucial for expanding our understanding of disease progression and evaluating drug efficacy. However, due to the diverse functions of different enzymes, currently available methods for comprehensive analysis of these events are limited. Here, we present an in situ track-generated DNA walker for AND-gate logic imaging of telomerase (TE) and flap endonuclease 1 (FEN1) activities in live cells. TE is in charge of generating the tracks for the walking strands by extending the TE primer on a gold nanoparticle, while FEN1 is responsible for recognizing the overlapping structure formed by the walking strands and the tracks and then cleaving the fluorescent reporter to produce signals. By utilizing the DNA walker, we successfully determined the expression levels and activities of TE and FEN1 in various cancer cell lines, offering promising prospects for screening inhibitors and investigating the biomolecular mechanisms of diseases.

A one-pot transcriptional assay method that detects the tumor biomarker FEN1 based on its flap cleavage activity.

BACKGROUND: Detection of tumor biomarkers in body fluids is a significant advancement in cancer treatment because it allows diagnosis without invasive tissue biopsies. Nucleases have long been regarded as a potential class of biomarkers that can indicate the occurrence and progression of cancers. Among these, flap endonuclease 1 (FEN1) plays an important role in DNA replication and repair, and also overexpressed in abnormally proliferating cells such as cancer cells. FEN1 is thus considered to be a potential biomarker as well as a target for cancer therapy. RESULTS: We developed a novel method for detecting FEN1 based on its specific endonuclease activity which incises bifurcated nucleic acids (flaps), in combination with in vitro transcription. Developed method uses a simple DNA structure (substrate DNA) carrying a short 5'-flap sequence, and a single-stranded sensor DNA encoding the Broccoli light-up aptamer. When the assay mixture was supplied with a FEN1-containing sample, the flap sequence encoding the sense sequence of T7 promoter was cleaved and released from the substrate DNA. Because the sensor DNA was designed to carry the Broccoli RNA aptamer under the antisense sequence of T7 promoter, hybridization of the excised flap onto the sensor DNA initiated the transcription of the Broccoli RNA aptamer, enabling determination of the FEN1 titer based on the fluorescence of transcribed Broccoli aptamer. By using a combination of FEN1-mediated generation of a short oligonucleotide and subsequent oligonucleotide-dependent in vitro transcription, this method could detect FEN1 in biological samples within 1 h. SIGNIFICANCE AND NOVELTY: Developed method enables the detection of FEN1 by a simple one-pot reaction. It can detect sub-nanomolar concentrations of FEN1 within an hour, and has the potential to be used for cancer diagnosis, prognosis, and drug screening. It also enables easy identification of compounds that inhibit FEN1 activity and is thus a versatile platform for screening anti-cancer drugs. We anticipate that the basic principles of this assay can be applied to detect other biomolecules, such as nucleic acids.

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.

FEN1 inhibitor SC13 promotes CAR-T cells infiltration into solid tumours through cGAS-STING signalling pathway.

It is well known that chimeric antigen receptor T-cell immunotherapy (CAR-T-cell immunotherapy) has excellent therapeutic effect in haematological tumours, but it still faces great challenges in solid tumours, including inefficient T-cell tumour infiltration and poor functional persistence. Flap structure-specific endonuclease 1 (FEN1), highly expressed in a variety of cancer cells, plays an important role in both DNA replication and repair. Previous studies have reported that FEN1 inhibition is an effective strategy for cancer treatment. Therefore, we hypothesized whether FEN1 inhibitors combined with CAR-T-cell immunotherapy would have a stronger killing effect on solid tumours. The results showed that low dose of FEN1 inhibitors SC13 could induce an increase of double-stranded broken DNA (dsDNA) in the cytoplasm. Cytosolic dsDNA can activate the cyclic GMP-AMP synthase-stimulator of interferon gene signalling pathway and increase the secretion of chemokines. In vivo, under the action of FEN1 inhibitor SC13, more chemokines were produced at solid tumour sites, which promoted the infiltration of CAR-T cells and improved anti-tumour immunity. These findings suggest that FEN1 inhibitors could enable CAR-T cells to overcome poor T-cell infiltration and improve the treatment of solid tumours.

Inhibition of FEN1 promotes DNA damage and enhances chemotherapeutic response in prostate cancer cells.

Prostate cancer (PCa) refers to epithelial malignancies occurring in prostate and is the most commonly diagnosed cancer among men. Flap structure-specific endonuclease 1 (FEN1) is one of the major base excise repair enzymes and is abnormally expressed in a variety of cancers, which contributes to cancer progression. Targeting FEN1 serves as a potent strategy for cancer therapy. However, how FEN1 acts on PCa cell proliferation and its role in chemotherapeutic response remain largely unknown. In this study, we show that knockdown of FEN1 by CRISPR/Cas9 system impedes the proliferation and migration of PCa cells. FEN1 Inhibitor SC13 induced DNA damage accumulation and further resulted in apoptosis of PCa cells. Furthermore, genetic knockdown of FEN1 or inhibition of FEN1 by SC13 promoted DNA damage and enhanced docetaxel (DTX)-induced chemotherapeutic response in PCa cells. Collectively, these findings demonstrate the importance of FEN1 in PCa cell proliferation and implicate FEN1 as a promising target for monotherapy or combination therapeutic strategy in PCa treatment.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.

Target-activated T7 transcription circuit-mediated multiple cycling signal amplification for monitoring of flap endonuclease 1 activity in cancer cells.

The structure-specific endonuclease flap endonuclease 1 (FEN1) is an essential functional protein in DNA replication and genome stability, and it has been identified as a promising biomarker and drug target for multiple cancers. Herein, we develop a target-activated T7 transcription circuit-mediated multiple cycling signal amplification platform for monitoring FEN1 activity in cancer cells. In the presence of FEN1, the flapped dumbbell probe is cleaved to generate a free 5' flap single-stranded DNA (ssDNA) with the 3'-OH terminus. The ssDNA can hybridize with the T7 promoter-bearing template probe to trigger the extension with the aid of Klenow fragment (KF) DNA polymerase. Upon the addition of T7 RNA polymerase, an efficient T7 transcription amplification reaction is initiated to produce abundant single-stranded RNAs (ssRNAs). The ssRNA can hybridize with a molecular beacon to form an RNA/DNA heteroduplex that can be selectively digested by DSN to generate an enhanced fluorescence signal. This method exhibits good specificity and high sensitivity with a limit of detection (LOD) of 1.75 × 10-6 U µL-1. Moreover, it can be applied for the screening of FEN1 inhibitors and the monitoring of FEN1 activity in human cells, holding great potential in drug discovery and clinical diagnosis.

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.

Metformin sensitises osteosarcoma to chemotherapy via the IGF-1R/miR-610/FEN1 pathway.

Metformin can enhance cancer cell chemosensitivity to anticancer drugs. IGF-1R is involved in cancer chemoresistance. The current study aimed to elucidate the role of metformin in osteosarcoma (OS) cell chemosensitivity modulation and identify its underlying mechanism in IGF-1R/miR-610/FEN1 signalling. IGF-1R, miR-610, and FEN1 were aberrantly expressed in OS and participated in apoptosis modulation; this effect was abated by metformin treatment. Luciferase reporter assays confirmed that FEN1 is a direct target of miR-610. Moreover, metformin treatment decreased IGF-1R and FEN1 but elevated miR-610 expression. Metformin sensitised OS cells to cytotoxic agents, while FEN1 overexpression partly compromised metformin's sensitising effects. Furthermore, metformin was observed to enhance adriamycin's effects in a murine xenograft model. Metformin enhanced OS cell sensitivity to cytotoxic agents via the IGF-1R/miR-610/FEN1 signalling axis, highlighting its potential as an adjuvant during chemotherapy.

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.

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

BACKGROUND: Flap endonuclease 1 (FEN1), well known for its structural-specific nuclease, possessing 5'-flap endonuclease and 5'-3' exonuclease activities, 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 interaction etc., and influences many biological processes. Our previous studies on integrated succinylome profiles found that succinylation and acetylation levels of FEN1 would change under different conditions. Succinylation at FEN1 Lys200 site results in the accumulation of damaged DNA and increased susceptibility to fork-stalling agents. The interplay with other forms of modification could affects its protein interaction affinity and thus contribute to genome stability. OBJECTIVE: This article studied the biological role of FEN1 by acyl modification in HeLa cells. METHOD: In order to explore the function of FEN1 acylation in cells, we mimicked the presence or absence of acetylation or succinylation by mutating key amino acids to glutamic acid and glutamine. We carried out a series of experiments including cell cycle, MTS, enzyme kinetics measurements, immunofluorescence and so on. RESULTS: The absence of acylation of FEN1 leads to the blocked cell cycle process and the reduced efficiency of FEN1 on its DNA substrates, affecting the interaction of FEN1 with both repair and replication related proteins and thus its role in the repair of DNA damage. CONCLUSION: We have verified acyl groups could modify Lys125, Lys252 and Lys254 of FEN1. Acylation level of these three is important for enzyme activity, cell proliferation and DNA damage response, thus contributing to genome stability.

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.

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.