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A lateral flow strip (LFS) is an ideal tool for point-of-care testing (POCT), but traditional LFSs cannot be used for multiplex detection. Herein, a multiplex and versatile LFS based on flap endonuclease 1 (FEN1)-induced steric hindrance change (FISH-LFS) is proposed. In this method, multiplex PCR coupled with cascade invasive reactions was employed to yield single-stranded flaps, which were target-specific but independent of target sequences. Then, the amplicons were applied for FISH-LFS, and the single-stranded flaps would be efficiently captured by the complementary LFS-probes at different test lines. As flaps were cleaved from the specially designed hairpin probes, competition among flaps and hairpin probes would occur in capturing the probes at test lines. We enabled the hairpin probes to flow through the test lines while the flaps to stay at the test lines by making use of the difference in steric hindrance between hairpin probes and flaps. The assay is able to detect as low as two copies of blood pathogens (HBV, HCV, and HIV), to pick up as low as 0.1% mutants from wild-type gDNA, and to genotype 200 copies of SARS-CoV-2 variants α and β within 75 min at a conventional PCR engine. As the method is free of dye, a portable PCR engine could be used for a cost-effective multiplex detection on site. Results using an ultrafast mobile PCR system for FISH-LFS showed that as fast as 30 min was achieved for detecting three pathogens (HBV, HCV, and HIV) in blood, very suitable for POCT of pathogen screening. The method is convenient in operation, simple in instrumentation, specific in genotyping, and very easy in setting up multiplex POCT assays.

Flap endonuclease 1 (FEN1) is a critical protein in DNA repair, genomic stability, and carcinogenesis. Functional polymorphisms in FEN1 promoter -69GA (rs174538) and 3'UTR 4150GT (rs4246215), have been associated with the susceptibility to several cancers, including lung, breast, esophageal, gastric, liver, colorectal, and gallbladder cancer, as well as glioma, endometriosis, and leukemia. However, the contribution of FEN1 variant genotypes to oral cancer has never been examined. Thus, we aimed to evaluate the contribution of FEN1 rs174538 and rs4246215 genotypes to oral cancer risk in Taiwan.The contribution of FEN1 genotypes to oral cancer risk was examined in 958 oral cancer patients and 958 age- and sex-matched healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).The percentages of GG, AG, and AA genotypes at FEN1 rs174538 were 34.8%, 46.0%, and 19.2% among oral cancer patients and 37.8%, 45.2%, and 17.0% among healthy controls (p for trend=0.2788). The genotypic percentages of FEN1 rs4246215 were 35.9%, 45.9%, and 18.2% among oral cancer patients and 37.6%, 45.1%, and 17.3% among healthy controls (p for trend=0.7315). Overall, FEN1 rs174538 and rs4246215 were not differently distributed between the oral cancer patient and healthy control groups. The allele frequency analysis confirmed that FEN1 rs174538 and rs4246215 were non-differentially distributed among case and control groups (OR=1.11 and 1.05, 95%CI=0.98-1.27 and 0.93-1.20, p=0.1074 and 0.4491, respectively).FEN1 may contribute to oral cancer risk determination via protein expression and/or post-transcription modification, but may not be a practical genetic marker.

The removal of RNA primers is essential for mitochondrial DNA (mtDNA) replication. Several nucleases have been implicated in RNA primer removal in human mitochondria, however, no conclusive mechanism has been elucidated. Here, we reconstituted minimal in vitro system capable of processing RNA primers into ligatable DNA ends. We show that human 5′-3′ exonuclease, EXOG, plays a fundamental role in removal of the RNA primer. EXOG cleaves short and long RNA-containing flaps but also in cooperation with RNase H1, processes non-flap RNA-containing intermediates. Our data indicate that the enzymatic activity of both enzymes is necessary to process non-flap RNA-containing intermediates and that regardless of the pathway, EXOG-mediated RNA cleavage is necessary prior to ligation by DNA Ligase III. We also show that upregulation of EXOG levels in mitochondria increases ligation efficiency of RNA-containing substrates and discover physical interactions, both in vitro and in cellulo, between RNase H1 and EXOG, Pol γA, Pol γB and Lig III but not FEN1, which we demonstrate to be absent from mitochondria of human lung epithelial cells. Together, using human mtDNA replication enzymes, we reconstitute for the first time RNA primer removal reaction and propose a novel model for RNA primer processing in human mitochondria.

Overexpression of Flap endonuclease 1 (FEN1) has been previously implicated in hepatocellular carcinoma (HCC), while its expression features and mechanisms remain unclear. In the current study, differential expression genes (DEGs) were screened in HCC tissues and normal liver tissues in 4 Gene Expression Omnibus (GEO) datasets. FEN1, one of the hub co-overexpressed genes, was further determined overexpressed in HCC tissues in TCGA, local HCC cohorts, and hepatocarcinogenesis model. In addition, high expression of FEN1 indicated poor prognosis of HCC patients. Loss-of-function and gain-of-function assays demonstrated that FEN1 enhanced the proliferation, cell cycle phage transition, migration/ invasion, therapy resistance, xenograft growth, and epithelial-mesenchymal transition (EMT) process of HCC cells. Mechanically, FEN1 could inactivate P53 signaling by preventing the ubiquitination and degradation of mouse double minute 2 (MDM2) via recruiting ubiquitin-specific protease 7 (USP7). Interfering USP7 with P22077 significantly reversed the malignant phenotypes activated by FEN1. In conclusion, this study suggests FEN1 as a robust prognostic biomarker and potential target for HCC.

How cells with DNA replication defects acquire mutations that allow them to escape apoptosis under environmental stress is a long-standing question. Here, we report that an error-prone Okazaki fragment maturation (OFM) pathway is activated at restrictive temperatures in rad27Δ yeast cells. Restrictive temperature stress activated Dun1, facilitating transformation of unprocessed 5′ flaps into 3′ flaps, which were removed by 3′ nucleases, including DNA polymerase δ (Polδ). However, at certain regions, 3′ flaps formed secondary structures that facilitated 3′ end extension rather than degradation, producing alternative duplications with short spacer sequences, such as pol3 internal tandem duplications. Consequently, little 5′ flap was formed, suppressing rad27Δ-induced lethality at restrictive temperatures. We define a stress-induced, error-prone OFM pathway that generates mutations that counteract replication defects and drive cellular evolution and survival.

During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIPN-term is released from PCNA and only PIPDBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation., This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H. and A.D.B.) and the Competitive Research Award Grant CRG8 URF/1/4036‐01‐01 (to S.M.H. and A.D.B.). We acknowledge The Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB), major funding from MRC (MC_PC_17136). We thank Christos Savva and T.J. Ragan (LISCB, University of Leicester) for their help in cryo-EM data collection and advice on data processing. This project has been carried out using the resources of CSUC.

The final steps of lagging strand synthesis induce maturation of Okazaki fragments via removal of the RNA primers and ligation. Iterative cycles between Polymerase δ (Polδ) and Flap endonuclease-1 (FEN1) remove the primer, with an intermediary nick structure generated for each cycle. Here, we show that human Polδ is inefficient in releasing the nick product from FEN1, resulting in non-processive and remarkably slow RNA removal. Ligase 1 (Lig1) can release the nick from FEN1 and actively drive the reaction toward ligation. These mechanisms are coordinated by PCNA, which encircles DNA, and dynamically recruits Polδ, FEN1, and Lig1 to compete for their substrates. Our findings call for investigating additional pathways that may accelerate RNA removal in human cells, such as RNA pre-removal by RNase Hs, which, as demonstrated herein, enhances the maturation rate ~10-fold. They also suggest that FEN1 may attenuate the various activities of Polδ during DNA repair and recombination.

Nucleic acid detection is crucial for monitoring diseases for which rapid, sensitive, and easy-to-deploy diagnostic tools are needed. CRISPR-based technologies can potentially fulfill this need for nucleic acid detection. However, their widespread use has been restricted by the requirement of a protospacer adjacent motif in the target and extensive guide RNA optimization. In this study, we developed FELICX, a technique that can overcome these limitations and provide a useful alternative to existing technologies. FELICX comprises flap endonuclease, Taq ligase and CRISPR-Cas for diagnostics (X) and can be used for detecting nucleic acids and single-nucleotide polymorphisms. This method can be deployed as a point-of-care test, as only two temperatures are needed without thermocycling for its functionality, with the result generated on lateral flow strips. As a proof-of-concept, we showed that up to 0.6 copies/μL of DNA and RNA could be detected by FELICX in 60 min and 90 min, respectively, using simulated samples. Additionally, FELICX could be used to probe any base pair, unlike other CRISPR-based technologies. Finally, we demonstrated the versatility of FELICX by employing it for virus detection in infected human cells, the identification of antibiotic-resistant bacteria, and cancer diagnostics using simulated samples. Based on its unique advantages, we envision the use of FELICX as a next-generation CRISPR-based technology in nucleic acid diagnostics.

The continued uncertainty of emerging infectious viral diseases has led to an extraordinary urgency to develop advanced molecular diagnostic tests that are faster, more reliable, simpler to use, and readily available than traditional methods. This study presents a system that can accurately and rapidly trace viral nucleic acids by employing flap endonuclease 1 (FEN1)-assisted specific DNA cleavage reactions and surface-enhanced Raman scattering (SERS)-based analysis. The designed Raman tag-labeled 5'- and 3'-flap provider DNA yielded structurally defined DNA substrates on magnetic nanoparticle surfaces when a target was present. The FEN1 enzyme subsequently processes the substrates formed

As a critical functional protein in DNA replication and genome stability, flap endonuclease 1 (FEN1) has been considered a promising biomarker and druggable target for multiple cancers. We report here a transcription-powered clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a signal expansion platform for rapid and sensitive detection of FEN1. In this method, the probe cleavage by FEN1 generated a free 5' flap single-stranded DNA which could hybridize with the single-stranded T7 promoter-bearing template and trigger the extension. Then, the CRISPR guide RNA (crRNA) transcribed from the extended template activated the collateral DNase activity of Cas12a, releasing the fluorophore from the quenched DNA signal probe to report the FEN1 detection result. The high specificity for FEN1 was validated by comparing with other repair-relevant proteins. The limit of detection (LOD) could be as low as 0.03 mU, which is sensitive enough to detect the FEN1 activity in biological samples. In addition, the inhibition assay of FEN1 was also successfully achieved with this platform, proving its potential in inhibitor screening. In summary, this study provides a novel biosensor for FEN1 activity analysis and provides new insights into the development of CRISPR-based biosensors for non-nucleic acid targets.

Chronic myeloid leukemia (CML) is characterized by the formation of the BCR-ABL fusion gene. The BCR-ABL protein leads to an increased level of reactive oxygen species, which is a major cause of endogenous DNA double-strand breaks (DSBs). CML cells are prone to rely on a highly mutagenic alternative end-joining (Alt-EJ) pathway to cope with enhanced DSBs, which aggravates chromosomal instability. Hence, targeting dysregulated DNA repair proteins provides new insights into cancer treatment. In this study, we discovered the abnormal upregulation of Flap endonuclease 1 (FEN1) in CML, as well as FEN1's participation in the error-prone Alt-EJ repair pathway and its interplay with DNA Ligase1 and proliferating cell nuclear antigen in DSB repair. Knockdown of FEN1 by shRNA not only inhibited the proliferation and induced apoptosis but also enhanced the efficacy of imatinib (IM) in drug-resistant CML cell K562/G01. Moreover, excessive DSB accumulation was detected after FEN1 inhibition. In summary, our results demonstrated that FEN1 is a promising therapeutic target in CML treatment. This work extends the understanding of regulating abnormal DSB repair for cancer treatment.

Mutagenesis is a multifactor process associated with increased risk of cancer. Polycyclic aromatic hydrocarbons (PAHs) exposure and genetic susceptibility were conductive to genotoxic effects including gene damage, which can increase mutational probability and are potential carcinogenic etiology. We aimed to explore the dose-effect associations of PAHs exposure with damage of exons of epidermal growth factor receptor (EGFR) and breast cancer susceptibility gene 1 (BRCA1), as well as their associations whether modified by Flap endonuclease 1 (FEN1) genotype. 288 coke oven male workers were recruited and we detected the concentration of 1-hydroxypyrene (1-OH-pyr) as PAHs exposure biomarker in urine and examined base modification in exons of EGFR and BRCA1 respectively, and genotyped FEN1 rs174538 polymorphism in plasma by PCR methods. Compared to low exposure group, the high exposed workers had significantly higher urinary concentrations of 1-OH-pyr, after adjustment for multiple covariates (P Ptrend < 0.001). The multiple linear regression analysis showed that the levels of urinary 1-OH-pyr were both significantly associated with increased EGFR-19 and EGFR-21 in both smokers and nonsmokers (both P P Pinteraction< 0.05). Our findings revealed the linear dose-effect association between exon damage of EGFR and PAHs exposure and highlight differences in genetic contributions to exon damage and have the potential to identify at-risk subpopulations who are susceptible to adverse health effects induced by PAH exposure.

Flap endonuclease 1 (FEN1) is an endonuclease that specially removes 5' single-stranded overhang of branched duplex DNA (5' flap). While FEN1 is essential in various DNA metabolism pathways for preventing the malignant transformation of cells, an unusual expression of FEN1 is often associated with tumor progression, making it a potential biomarker for cancer diagnosis and treatment. Here we report a multimodal detection of FEN1 activity based on CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides (ssDNA). A dumbbell DNA structure with a 5' flap was designed, which can be cleaved by the FEN1 and the dumbbell DNA is subsequently ligated by T4 DNA ligase. The resulting closed duplex DNA contains a specific protospacer adjacent motif (PAM) that activates trans-cleavage of ssDNA after binding to CRISPR/Cas12a-crRNA. The trans-cleavage is activated only once and is independent to length or sequence of the ssDNA, which allows efficient signal amplification and multimodal signals such as fluorescence or cleaved connection between magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that alters solution turbidity after magnetic separation. In addition, by loading the particle solution into a microfluidic chip, unconnected PMPs escaping from a magnetic separator are amassed at the particle dam, enabling a visible PMP accumulation length proportional to the FEN1 activity. This multimodal detection is selective to FEN1 and achieves a low limit of detection (LOD) with only 40 min of reaction time. Applying to cell lysates, higher FEN1 activity was detected in breast cancer cells, suggesting a great potential for cancer diagnosis.

Specific and sensitive detection of flap endonuclease 1 (FEN1), an enzyme biomarker involved in DNA replications and several metabolic pathways, is of high values for the diagnosis of various cancers. In this work, a fluorescence strategy based on transcriptional amplification of lighting-up aptamers for label-free, low background and sensitive monitoring of FEN1 is developed. FEN1 cleaves the 5' flap of the DNA complex probe with double flaps to form a notched dsDNA, which is ligated by T4 DNA ligase to yield fully complementary dsDNA. Subsequently, T7 RNA polymerase binds the promoter region to initiate cyclic transcriptional generation of many RNA aptamers that associate with the malachite green dye to yield highly amplified fluorescence for detecting FEN1 with detection limit as low as 0.22 pM in a selective way. In addition, the method can achieve diluted serum monitoring of low concentrations of FEN1, exhibiting its potential for the diagnosis of early-stage cancers.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that cleaves the 5' single-stranded protrusion (also known as 5' flap) during Okazaki fragment processing. It is overexpressed in various types of human cancer cells and has been considered as an important biomarker for cancer diagnosis. However, conventional methods for FEN1 assay usually suffer from complicated platform and laborious procedures with a limited sensitivity. Here, we developed a dual-signal method for sensitive detection of FEN1 on the basis of duplex-specific nuclease actuated cyclic enzymatic repairing-mediated signal amplification. Once the 5' flap of the double-flap DNA substrate was cleaved by target FEN1, the cleaved 5' flap initiated strand-displacement amplification to produce plenty of G-rich DNA (G) sequences. These G sequences that self-assembled into G-quadruplexes in the presence of hemin revealed horseradish-peroxidase-like catalytic activities as well as fluorescence enhancement of thioflavin T. The UV-vis signal showed a good linear relationship with the logarithm of FEN1 activity ranging from 0.03 to 1.5 U with a detection limit of 0.01 U. The fluorescence signal correlated linearly with the logarithm of FEN1 activity ranging from 0.001 to 1.5 U with a detection limit of 0.75 mU. In addition, FEN1 can be visualized not only by colorimetry but also by fluorescence (under ice-water mixture conditions). This reliable, accurate, and convenient method would be a potential powerful tool in point-of-care testing applications and therapeutic response assessment.

Background Flap endonuclease 1 (FEN1) is a structure-specific nuclease that plays a role in a variety of DNA metabolism processes. FEN1 is important for maintaining genomic stability and regulating cell growth and development. It is associated with the occurrence and development of several diseases, especially cancers. There is a lack of systematic bibliometric analyses focusing on research trends and knowledge structures related to FEN1. Purpose To analyze hotspots, the current state and research frontiers performed for FEN1 over the past 15 years. Methods Publications were retrieved from the Web of Science Core Collection (WoSCC) database, analyzing publication dates ranging from 2005 to 2019. VOSviewer1.6.15 and Citespace5.7 R1 were used to perform a bibliometric analysis in terms of countries, institutions, authors, journals and research areas related to FEN1. A total of 421 publications were included in this analysis. Results Our findings indicated that FEN1 has received more attention and interest from researchers in the past 15 years. Institutes in the United States, specifically the Beckman Research Institute of City of Hope published the most research related to FEN1. Shen BH, Zheng L and Bambara Ra were the most active researchers investigating this endonuclease and most of this research was published in the Journal of Biological Chemistry. The main scientific areas of FEN1 were related to biochemistry, molecular biology, cell biology, genetics and oncology. Research hotspots included biological activities, DNA metabolism mechanisms, protein-protein interactions and gene mutations. Research frontiers included oxidative stress, phosphorylation and tumor progression and treatment. Conclusion This bibliometric study may aid researchers in the understanding of the knowledge base and research frontiers associated with FEN1. In addition, emerging hotspots for research can be used as the subjects of future studies.

Camptothecin has been used in tumor therapy for a long time but its antitumor effect is rather limited due to the side effect and the drug resistance. FEN1, a major component of DNA repair systems, plays important roles in maintaining genomic stability via DNA replication and repair. Here we found that FEN1 inhibitor greatly sensitizes cancer cells to low-dose camptothecin. The combinative treatment of FEN1 inhibitor and 1 nM camptothecin induced a synthetic lethal effect, which synergistically suppressed cancer cell proliferation and significantly mediated apoptosis both in vitro and in vivo. Our study suggested that targeting FEN1 could be a potent strategy for tumor-targeting cancer therapy.

DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM., DNA ligase 1 (LIG1) finalizes eukaryotic nuclear DNA synthesis by sealing Okazaki fragments using DNA end-joining reactions. Here the authors, by studying an engineered low-fidelity LIG1, reveal that LIG1 is a highly accurate DNA ligase in vivo.

A flap endonuclease 1 (FEN 1)-assisted swing arm DNA walker was constructed to achieve the signal amplification detection of ctDNA. The MB-labeled hairpin DNA was designed as the track and a long swing-arm DNA strand as the capture probe. The introduction of ctDNA unlocked a helper hairpin DNA, which could be captured to form the DNA duplex walker with the capture probe, and also activated the catalyst hairpin assembly. The DNA duplex walker opened the hairpin track and formed a three-base overlapping DNA structure, which was recognized and cleaved by FEN 1. Driven by the FEN 1 and the high reaction temperature, the DNA walker was initiated to hybridize with the track DNA and release multiple MB-labeled flaps for signal amplification. Owing to the excellent amplification capacity of the target recycling-induced DNA walker and programmed catalysis hairpin assembly, the one-step biosensor showed a linear detection range from 1 fM to 100 pM with a detection limit of 0.33 fM. Moreover, the sensitive detection of ctDNA in serum samples was verified, suggesting its potential application in liquid biopsy for clinical diagnosis.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease, which catalyzes the removal of 5' overhanging DNA flap from a specific DNA structure. FEN1 has been considered as an important biomarker for cancer diagnosis since it is over-expressed in various types of human tumor cells and closely related to cancer development. Nanoprobes gradually become basic tools for analyzing biomarkers variations in vivo. Here, we utilized aminoated mesoporous silica nanoparticles (NH

A sensitive homogeneous electrochemiluminescence (ECL) biosensor for flap endonuclease 1 (FEN1) detection was developed by combining highly sensitive ECL detection, high efficiency of branched hybridization chain reaction (BHCR) amplification, a convenient homogeneous strategy, and simple ultrafiltration separation. Magnetic beads were first modified with well-designed double flap DNAs containing 5'-flaps. In the presence of FEN1, the 5'-flap can be cleaved, and a large amount of single-stranded DNA can be produced, which can be separated easily from the double-flap DNA-modified beads by a magnet. Then, the cleaved 5'-flap can be used to initiate BHCR amplification to produce a large amount of long-strand dsDNA. Ru(phen)

Solar ultraviolet radiation (UV) can cause DNA damage in microorganisms. Flap endonuclease 1 (FEN1) is a structure-specific nuclease and plays important roles in DNA replication and repair. At present, the properties and functions of FEN1 have not been characterized in detail in invertebrates such as Bombyx mori. In this study, Bombyx mori FEN1 (BmFEN1) was expressed in E. coli, and was shown to have nuclease activity that nonspecifically cleaved DNA in vitro. However, inside the cell, BmFEN1 did not cleave DNA randomly. Truncated BmFEN1 missing the nuclear localization signal (346-380 aa) still had the nuclease activity, but was no longer precisely localized to the sites of UV-induced DNA damage. It was further found that BmFEN1 favored the faster repair of UV-damaged DNA. The present study will provide a reference for further understanding the functions of BmFEN1 and UV-induced DNA damage repair mechanisms in insects.

Ovarian cancer (OC) is one of the most lethal gynecological malignancies in the world. The aim of the present study was to examine the role of microRNA (miR)-134-3p in OC. Reverse transcription-quantitative PCR was used to measure the expression levels of miR-134-3p. Cell Counting Kit-8, TUNEL, flow cytometric and colony formation assays were performed to examine the effects of miR-134-3p on OC cell proliferation. Moreover, wound healing and Transwell assays were performed to examine the effects on migration and invasion. In addition, western blot analyses were used to assess protein expression. Finally, the target genes of miR-134-3p were analyzed by bioinformatics analysis and Dual-Luciferase reporter assay. The results revealed that miR-134-3p expression was low in OC cells compared with in normal ovarian cells. The overexpression of miR-134-3p decreased cell viability, facilitated cell apoptosis, inhibited cell proliferation and arrested the cell cycle in SKOV-3 and OVCAR-3 cells. Furthermore, transfection using a miR-134-3p mimic inhibited the migration and invasion of SKOV-3 and OVCAR-3 cells, and decreased the protein expression levels of cyclooxygenase-2, matrix metalloproteinase (MMP)2 and MMP9. Bioinformatics analysis indicated that one of the potential target genes of miR-134-3p was flap structure-specific endonuclease 1 (FEN1), which was confirmed by dual-luciferase reporter assay. Moreover, overexpression of miR-134-3p decreased the expression levels of FEN1 in SKOV-3 and OVCAR-3 cells. Additionally, overexpression of FEN1 reversed the effects of the miR-134-3p mimic on the proliferation, migration and invasion of SKOV-3 and OVCAR-3 cells. Overall, the findings of the present study demonstrated that miR-134-3p may inhibit OC cell proliferation, migration and invasion by directly targeting FEN1.

The Saccharomyces cerevisiae Mus81-Mms4 complex is a highly conserved DNA structure-specific endonuclease that plays essential roles in the processing of recombination intermediates that arise during the repair of stalled replication forks and double-stranded breaks. To identify novel factors functioning conjointly with Mus81-Mms4, we performed a biochemical screen and found that Crp1, a cruciform DNA-recognizing protein that specifically binds to DNA four-way junction structures, could stimulate the Mus81-Mms4 endonuclease. The specific protein interaction between Mus81-Mms4 and Crp1 was responsible for the stimulation observed. Multicopy expression of Crp1 could partially rescue the sensitivity to DNA-damaging agents of the sgs1∆mus81∆21-24N mutant. Our results provide insight into the functional role and interaction of Crp1 with other proteins involved in DNA repair.

Human flap endonuclease 1 (FEN1) is a structure-specific, multifunctional endonuclease essential for DNA replication and repair. Our previous study showed that in response to DNA damage, FEN1 interacts with the PCNA-like Rad9-Rad1-Hus1 complex instead of PCNA to engage in DNA repair activities, such as stalled DNA replication fork repair, and undergoes SUMOylation by SUMO-1. Here, we report that succinylation of FEN1 was stimulated in response to DNA replication fork-stalling agents, such as ultraviolet (UV) irradiation, hydroxyurea, camptothecin, and mitomycin C. K200 is a key succinylation site of FEN1 that is essential for gap endonuclease activity and could be suppressed by methylation and stimulated by phosphorylation to promote SUMO-1 modification. Succinylation at K200 of FEN1 promoted the interaction of FEN1 with the Rad9-Rad1-Hus1 complex to rescue stalled replication forks. Impairment of FEN1 succinylation led to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of FEN1 succinylation in regulating its roles in DNA replication and repair, thus maintaining genome stability.

Flap endonuclease‑1 (FEN1), a structure‑specific nuclease participating in DNA replication and repair processes, has been confirmed to promote the proliferation and drug resistance of tumor cells. However, the biological functions of FEN1 in cancer cell migration and invasion have not been defined. In the present study, using online database analysis and immunohistochemistry of the specimens, it was found that FEN1 expression was associated with a highly invasive triple‑negative breast cancer (TNBC) subtype in both breast cancer samples from the Oncomine database and from patients recruited into the study. Furthermore, FEN1 was an important biomarker of lymph node metastasis and poor prognosis in patients with TNBC. FEN1 promoted migration of TNBC cell lines and FEN1 knockdown reduced the number of spontaneous lung metastasis in vivo. Ingenuity Pathway Analysis of FEN1‑related transcripts in 198 patients with TNBC demonstrated that the polo‑like kinase family may be the downstream target of FEN1. PLK4 was further identified as a critical target of FEN1 mediating TNBC cell migration, by regulating actin cytoskeleton rearrangement. The results of the present study validate FEN1 as a therapeutic target in patients with TNBC and revealed a new role for FEN1 in regulating TNBC invasion and metastasis.

Trinucleotide repeat (TNR) expansion and deletion are responsible for over 40 neurodegenerative diseases and associated with cancer. TNRs can undergo somatic instability that is mediated by DNA damage and repair and gene transcription. Recent studies have pointed toward a role for R-loops in causing TNR expansion and deletion, and it has been shown that base excision repair (BER) can result in CAG repeat deletion from R-loops in yeast. However, it remains unknown how BER in R-loops can mediate TNR instability. In this study, using biochemical approaches, we examined BER enzymatic activities and their influence on TNR R-loops. We found that AP endonuclease 1 incised an abasic site on the nontemplate strand of a TNR R-loop, creating a double-flap intermediate containing an RNA:DNA hybrid that subsequently inhibited polymerase β (pol β) synthesis of TNRs. This stimulated flap endonuclease 1 (FEN1) cleavage of TNRs engaged in an R-loop. Moreover, we showed that FEN1 also efficiently cleaved the RNA strand, facilitating pol β loop/hairpin bypass synthesis and the resolution of TNR R-loops through BER. Consequently, this resulted in fewer TNRs synthesized by pol β than those removed by FEN1, thereby leading to repeat deletion. Our results indicate that TNR R-loops preferentially lead to repeat deletion during BER by disrupting the balance between the addition and removal of TNRs. Our discoveries open a new avenue for the treatment and prevention of repeat expansion diseases and cancer.

Cervical cancer (CC) is a frequently occurring cancer in women with a high mortality rate. Despite improvements to therapeutic strategies, the survival outcome for patients with CC remains poor. Therefore, the present study aimed to investigate the molecular mechanism underlying CC inhibition involving microRNA (miR)‑140‑5p and flap structure‑specific endonuclease 1 (FEN1). Bioinformatics analysis was conducted, which identified that FEN1 was associated with CC cell cycle progression. Subsequently, 3'untranslated region reporter assays were performed to assess the regulatory relationship between FEN1 mRNA and miR‑140‑5p. Functional assays, including EdU staining assay, flow cytometry, and wound healing assays, were conducted to observe CC cell phenotypes induced by alterations to miR‑140‑5p and FEN1 expression levels. FEN1 expression was high and miR‑140‑5p expression was low in CC tissues and cell lines compared with adjacent healthy tissues and a normal cervical epithelial cell line, respectively. miR‑140‑5p knockdown reversed small interfering RNA‑FEN1‑mediated suppressive effects on CC cell phenotypes, potentially via inducing cell cycle arrest at the G1 phase. Therefore, the present study suggested that miR‑140‑5p may serve as an antitumorigenesis factor in CC by targeting FEN1 mRNA.

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor and has the highest mortality rate among cancers and high resistance to radiation and cytotoxic chemotherapy. Although some targeted therapies can partially inhibit oncogenic mutation-driven proliferation of GBM cells, therapies harnessing synthetic lethality are ‘coincidental’ treatments with high effectiveness in cancers with gene mutations, such as GBM, which frequently exhibits DNA-PKcs mutation. By implementing a highly efficient high-throughput screening (HTS) platform using an in-house-constructed genome-wide human microRNA inhibitor library, we demonstrated that miR-1193 inhibition sensitized GBM tumor cells with DNA-PKcs deficiency. Furthermore, we found that miR-1193 directly targets YY1AP1, leading to subsequent inhibition of FEN1, an important factor in DNA damage repair. Inhibition of miR-1193 resulted in accumulation of DNA double-strand breaks and thus increased genomic instability. RPA-coated ssDNA structures enhanced ATR checkpoint kinase activity, subsequently activating the CHK1/p53/apoptosis axis. These data provide a preclinical theory for the application of miR-1193 inhibition as a potential synthetic lethal approach targeting GBM cancer cells with DNA-PKcs deficiency.

Given the broad utility of humanized yeast to model and study human biology, a reference set of human genes that can replace cognate yeast genes and operate in yeast is needed. Hamza et al. present..., Cross-species complementation can be used to generate humanized yeast, which is a valuable resource with which to model and study human biology. Humanized yeast can be used as an in vivo platform to screen for chemical inhibition of human protein drug targets. To this end, we report the systematic complementation of nonessential yeast genes implicated in chromosome instability (CIN) with their human homologs. We identified 20 human–yeast complementation pairs that are replaceable in 44 assays that test rescue of chemical sensitivity and/or CIN defects. We selected a human–yeast pair (hFEN1/yRAD27), which is frequently overexpressed in cancer and is an anticancer therapeutic target, to perform in vivo inhibitor assays using a humanized yeast cell-based platform. In agreement with published in vitro assays, we demonstrate that HU-based PTPD is a species-specific hFEN1 inhibitor. In contrast, another reported hFEN1 inhibitor, the arylstibonic acid derivative NSC-13755, was determined to have off-target effects resulting in a synthetic lethal phenotype with yRAD27-deficient strains. Our study expands the list of human–yeast complementation pairs to nonessential genes by defining novel cell-based assays that can be utilized as a broad resource to study human drug targets.