Search

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3'-5' exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.

Purpose: Metastasis of hepatocellular carcinoma (HCC) critically impacts the survival prognosis of patients, with the pivotal role of hepatocellular carcinoma stem cells in initiating invasive metastatic behaviors. The Flap Endonuclease 1 (FEN1) is delineated as a metallonuclease, quintessential for myriad cellular processes including DNA replication, DNA synthesis, DNA damage rectification, Okazaki fragment maturation, baseexcision repair, and the preservation of genomic stability. Furthermore, it has been recognized as an oncogene in a diverse range of malignancies. Our antecedent research has highlighted a pronounced overexpression of protein FEN1 in hepatocellular carcinoma, where it amplifies the invasiveness and metastatic potential of liver cancer cells. However, its precise role in liver cancer stem cells (LCSCs) remains an enigma and requires further investigation. Methods: To rigorously evaluate the stemness attributes of LCSCs, we employed sphere formation assays and flow cytometric evaluations. Both CD133+ and CD133- cell populations were discerningly isolated utilizing immunomagnetic bead separation techniques. The expression levels of pertinent genes were assayed via real-time quantitative PCR (RT-qPCR) and western blot analyses, while the expression profiles in hepatocellular carcinoma tissues were gauged using immunohistochemistry. Subsequent immunoprecipitation, in conjunction with mass spectrometry, ascertained the concurrent binding of proteins FEN1 and Small ubiquitin-related modifier 2 (SUMO2) in HCC cells. Lastly, the impact of SUMO2 on proteasomal degradation pathway of FEN1 was validated by supplementing MG132. Results: Our empirical findings substantiate that protein FEN1 is profusely expressed in spheroids and CD133+ cells. In vitro investigations demonstrate that the upregulation of protein FEN1 unequivocally augments the stemness of LCSCs. In a congruent in vivo context, elevation of FEN1 noticeably enhances the tumorigenic potential of LCSCs. Conversely, inhibiting protein FEN1 resulted in a marked reduction in LCSC stemness. From a mechanistic perspective, there exists a salient positive correlation between the protein expression of FEN1 and SUMO2 in liver cancer tissues. Furthermore, the level of SUMO2-mediated modification of FEN1 is pronouncedly elevated in LCSCs. Interestingly, SUMO2 has the ability to bind to FEN1, leading to a inhibition in the proteasomal degradation pathway of FEN1 and an enhancement in its protein expression. However, it is noteworthy that this interaction does not affect the mRNA level of FEN1. Conclusion: In summation, our research elucidates that protein FEN1 is an effector in augmenting the stemness of LCSCs. Consequently, strategic attenuation of protein FEN1 might proffer a pioneering approach for the efficacious elimination of LCSCs.

Abstract 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.

Single-nucleotide polymorphism (SNP) plays a key role in the carcinogenesis of the human genome, and understanding the intrinsic relationship between individual genetic variations and carcinogenesis lies heavily in the establishment of a precise and sensitive SNP detection platform. Given this, a powerful and reliable SNP detection platform is proposed by a flap endonuclease 1 (FEN 1)-driven DNA walker-like reaction coupling with a magnetic bead (MB)-based separation. A carboxyfluorescein (FAM)-labeled downstream probe (DP) was decorated on a streptavidin magnetic bead (SMB). The target DNA, as a walker strand, was captured by hybridization with DP and an upstream probe (UP) to form a three-base overlapping structure and execute the walking function on the surface of SMB. FEN 1 was employed to specifically recognize the three-base overlapping structure and cut the 5′flap at the SNP site to report the walking event and signal amplification. Considering the fact that the fluorescence was labeled on the cleavage and uncleavage sequences of DP and the target DNA-triggered walking event was undistinguishable from the mixtures, magnetic separation came in handy for cleavage probe (CP) isolation and discrimination of the amplified signal from the background signal. In comparison with the conventional DNA walker reaction, this strategy was coupling with SMB-based separation, thus promising a powerful and reliable method for SNP detection and signal amplification.

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. [ABSTRACT FROM AUTHOR]

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that can specially identify and cleave 5′ flap of branched duplex DNA, and it plays a critical role in DNA metabolic pathways and human diseases. Herein, we propose a simple "mix-and-detection" strategy for sensitive measurement of human cellular FEN1 on basis of template-free amplification. We design a dumbbell probe with 5′ flap as a substrate of FEN1 and a NH 2 -labeled 3′ termini to prevent nonspecific amplification. When FEN1 is present, the 5′ flap is cleaved to release a free 3′-OH termini, initiating Ribonuclease HII (RNase HII)-assisted terminal deoxynucleotidyl transferase (TdT)-induced amplification for the production of a significant fluorescence signal. Due to the high exactitude of TdT-mediated extension reaction and RNase HII-induced single ribonucleotide excise, this assay shows excellent specificity and high sensitivity with a detection limit of 5.64 × 10−6 U/μL. Importantly, it can detect intracellular FEN1 activity with single-cell sensitivity under isothermal condition in a "mix-and-detection" manner, screen the FEN1 inhibitors, and even discriminate tumor cells from normal cells, offering a new platform for disease diagnosis and drug discovery. We develop a simple "mix-and-detection" strategy for sensitive measurement of human cellular FEN1 on basis of template-free amplification. [Display omitted] • We develop a simple "mix-and-detection" strategy for cellular FEN1 assay. • This assay shows excellent specificity and high sensitivity. • This method can detect intracellular FEN1 activity with single-cell sensitivity. • This method can screen the FEN1 inhibitors. • This method can discriminate tumor cells from normal cells. [ABSTRACT FROM AUTHOR]

Rapid and accurate diagnostic methods are crucial for managing viral gastroenteritis in children, a leading cause of global childhood morbidity and mortality. This study introduces a novel microfluidic-Flap endonuclease 1 (FEN1)-assisted isothermal amplification (MFIA) method for simultaneously detecting major viral pathogens associated with childhood diarrhea—rotavirus, norovirus, and adenovirus. Leveraging the specificity-enhancing properties of FEN1 with a universal dspacer-modified flap probe and the adaptability of microfluidic technology, MFIA demonstrated an exceptional detection limit (5 copies/μL) and specificity in the simultaneous detection of common diarrhea pathogens in clinical samples. Our approach addresses the limitations of current diagnostic techniques by offering a rapid (turn around time <1 h), cost-effective, easy design steps (universal flap design), and excellent detection performance method suitable for multiple applications. The validation of MFIA against the gold-standard PCR method using 150 actual clinical samples showed no statistical difference in the detection performance of the two methods, positioning it as a potential detection tool in pediatric diagnostic virology and public health surveillance. In conclusion, the MFIA method promises to transform pediatric infectious disease diagnostics and contribute significantly to global health efforts combating viral gastroenteritis. [ABSTRACT FROM AUTHOR]

To achieve highly sensitive and reliable detection of apurinic/apyrimidinic endonuclease 1 (APE1), a critical cancer diagnostic biomarker, we designed a DNA walker-based dual-mode biosensor, utilizing cellular endogenous dual enzymes (APE 1 and Flap endonuclease 1 (FEN 1)) to collaborate in activating and propelling DNA walker motion on DNA-functionalized Au nanoparticles. Incorporating both fluorescence and electrochemical detection modes, this system leverages signal amplification from DNA walker movement and cascade amplification through tandem hybridization chain reactions (HCR), achieving highly sensitive detection of APE 1. In the fluorescence mode, continuous DNA walker movement, initiated by APE1 and driven by FEN1, generates a robust signal response within a concentration range of 0.01–500 U mL−1, presenting a good linearity in the concentration range of 0.01–10 U mL−1, with a detection limit of 0.01 U mL−1. In the electrochemical detection module, the cascade upstream DNA walker and downstream HCR dual signal amplification strategy further enhances the sensitivity of APE1 detection, extending the linear range to 0.01–50 U mL−1 and reducing the detection limit to 0.002 U mL−1. Rigorous validation demonstrates the biosensor's specificity and anti-interference capability against multiple enzymes. Moreover, it effectively distinguishes cancer cells from normal cell lysates, exhibiting excellent stability and consistency in the dual-modes. Overall, our findings underscore the efficacy of the developed dual-mode biosensor for detecting APE1 in serum and cell lysates samples, indicating its potential for clinical applications in disease diagnosis. [ABSTRACT FROM AUTHOR]

Target-induced differences in the electrostatic interactions between methylene blue (MB) and indium tin oxide (ITO) electrode surface was firstly employed to develop a homogeneous electrochemical biosensor for flap endonuclease 1 (FEN1) detection. In the absence of FEN1, the positively charged methylene blue (MB) is free in the solution and can diffuse onto the negatively charged ITO electrode surface easily, resulting in an obvious electrochemical signal. Conversely, with the presence of FEN1, a 5′-flap is cleaved from the well-designed flapped dumbbell DNA probe (FDP). The remained DNA fragment forms a closed dumbbell DNA probe to trigger hyperbranched rolling circle amplification (HRCA) reaction, generating plentiful dsDNA sequences. A large amount of MB could be inserted into the produced dsDNA sequences to form MB-dsDNA complexes, which contain a large number of negative charges. Due to the strong electrostatic repulsion between MB-dsDNA complexes and the ITO electrode surface, a significant signal drop occurs. The signal change (ΔCurrent) shows a linear relationship with the logarithm of FEN1 concentration from 0.04 to 80.0 U/L with a low detection limit of 0.003 U/L (S/N = 3). This study provides a label-free and homogeneous electrochemical platform for evaluating FEN1 activity. [ABSTRACT FROM AUTHOR]

Abstract 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.

Purpose: The aim of this research was to investigate the clinical significance of the expression of flap structure-specific endonuclease 1 (FEN1) in primary osteosarcoma. Methods: The expression of FEN1 was detected by immunohistochemistry analysis. The association of the expression of FEN1 in osteosarcoma with clinicopathological parameters was analyzed by using χ2 test or Fisher's exact test. Survival analyses were performed by Kaplan–Meier method and Cox proportional hazards regression model. Results: Of the 40 osteosarcoma patients, 19 (47.5%) patients presented with FEN1 high expression, while in the non-neoplastic bone specimens, the FEN1 high expression was observed in 10% (3/30), the positive expression rate in osteosarcoma patients was significantly higher than that of non-neoplastic bone specimens (P< 0.01). Univariate analysis indicated that the progression-free survival (PFS) and overall survival (OS) were correlated with the expression level of FEN1 (PFS, P < 0.001; OS, P = 0.002), Enneking staging (PFS, P = 0.026; OS, P = 0.044) and chemotherapy response (PFS, P = 0.019; OS, P = 0.031). Multivariate analysis demonstrated that FEN1 expression was an independent prognostic factor for the PFS (HR = 4.73, P = 0.002) and OS (HR = 4.01, P = 0.038) of osteosarcoma patients. Conclusion: This study showed that FEN1 was overexpressed in osteosarcoma patients and positively associated with poor prognosis of osteosarcoma patients. Further studies should focus on the relative mechanisms and the targeted FEN1 therapies for osteosarcoma. [ABSTRACT FROM AUTHOR]

Target-induced differences in the electrostatic interactions between methylene blue (MB) and indium tin oxide (ITO) electrode surface was firstly employed to develop a homogeneous electrochemical biosensor for flap endonuclease 1 (FEN1) detection. In the absence of FEN1, the positively charged methylene blue (MB) is free in the solution and can diffuse onto the negatively charged ITO electrode surface easily, resulting in an obvious electrochemical signal. Conversely, with the presence of FEN1, a 5′-flap is cleaved from the well-designed flapped dumbbell DNA probe (FDP). The remained DNA fragment forms a closed dumbbell DNA probe to trigger hyperbranched rolling circle amplification (HRCA) reaction, generating plentiful dsDNA sequences. A large amount of MB could be inserted into the produced dsDNA sequences to form MB-dsDNA complexes, which contain a large number of negative charges. Due to the strong electrostatic repulsion between MB-dsDNA complexes and the ITO electrode surface, a significant signal drop occurs. The signal change (ΔCurrent) shows a linear relationship with the logarithm of FEN1 concentration from 0.04 to 80.0 U/L with a low detection limit of 0.003 U/L (S/N = 3). This study provides a label-free and homogeneous electrochemical platform for evaluating FEN1 activity.

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Bi 2 O 2 S 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 Bi 2 O 2 S 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. Synopsis: A PEC sensing platform based on Bi 2 O 2 S nanosheets has been constructed, which utilizes target mediated rolling ring amplification reaction to regulate the release of dopamine. Dopamine binds to the surface of Bi 2 O 2 S in situ to form surface defects and complete signal transduction, thereby realizing the facile and high-throughput detection of target FEN1. [Display omitted] • Dopamine coordination generates oxygen vacancies in situ on the surface of Bi 2 O 2 S nanosheets. • A label-free, immobilization-free, and high-throughput PEC sensing platform for FEN1 detection was developed. • The FEN1 was detected with a low detection limit of 1.42 × 10−4 U/mL. • The first report on the PEC detection of FEN1. • A new perspective was provided to explore Bi 2 O 2 S materials as an efficient PEC signal transducer. [ABSTRACT FROM AUTHOR]

DNA-modifying enzymes act as critical regulators in a wide range of genetic functions (e.g., DNA damage & repair, DNA replication), and their aberrant expression may interfere with regular genetic functions and induce various malignant diseases including cancers. DNA-modifying enzymes have emerged as the potential biomarkers in early diagnosis of diseases and new therapeutic targets in genomic research. Consequently, the development of highly specific and sensitive biosensors for the detection of DNA-modifying enzymes is of great importance for basic biomedical research, disease diagnosis, and drug discovery. Single-molecule fluorescence detection has been widely implemented in the field of molecular diagnosis due to its simplicity, high sensitivity, visualization capability, and low sample consumption. In this paper, we summarize the recent advances in single-molecule counting-based biosensors for DNA-modifying enzyme (i.e, alkaline phosphatase, DNA methyltransferase, DNA glycosylase, flap endonuclease 1, and telomerase) assays in the past four years (2019 − 2023). We highlight the principles and applications of these biosensors, and give new insight into the future challenges and perspectives in the development of single-molecule counting-based biosensors. [Display omitted] • We review single-molecule counting-based biosensors for DNA-modifying enzyme detection. • The principles and applications of these biosensors are highlighted. • The performances of different biosensors are compared. • We discuss the future challenges and perspectives in this field. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a common yet deadly form of malignant cancer. However, the specific mechanisms involved in HCC diagnosis have not yet fully elucidated. Herein, we screened four publically available Gene Expression Omnibus (GEO) expression profiles (GSE14520, GSE29721, GSE45267 and GSE60502), and used them to identify 409 differentially expressed genes (DEGs), including 142 and 267 up- and down-regulated genes, respectively. The DAVID database was used to look for functionally enriched pathways among DEGs, and the STRING database and Cytoscape platform were used to generate a protein-protein interaction (PPI) network for these DEGs. The cytoHubba plug-in was utilized to detect 185 hub genes, and three key clustering modules were constructed with the MCODE plug-in. Gene functional enrichment analyses of these three key clustering modules were further performed, and nine core genes including BIRC5, DLGAP5, DTL, FEN1, KIAA0101, KIF4A, MCM2, MKI67, and RFC4, were identified in the most critical cluster. Subsequently, the hierarchical clustering and expression of core genes in TCGA liver cancer tissues were analyzed using the UCSC Cancer Genomics Browser, and whether elevated core gene expression was linked to a poor prognosis in HCC patients was assessed using the GEPIA database. The PPI of the nine core genes revealed an interaction between FEN1, MCM2, RFC4, and BIRC5. Furthermore, the expression of FEN1 was positively correlated with that of three other core genes in TCGA liver cancer tissues. FEN1 expression in HCC and other tumor types was assessed with the FIREBROWSE and ONCOMINE databases, and results were verified in HCC samples and hepatoma cells. FEN1 levels were also positively correlated with tumor size, distant metastasis and vascular invasion. In conclusion, we identified nine core genes associated with HCC development, offering novel insight into HCC progression. In particular, the aberrantly elevated FEN1 may represent a potential biomarker for HCC diagnosis and treatment.

Flap endonuclease 1 (FEN1) is recognized as a pivotal factor in DNA replication, long-patch excision repair, and telomere maintenance. Excessive FEN1 expression has been reported to be closely associated with cancer progression, but the specific mechanism has not yet been explored. In the present study, we demonstrated that FEN1 promoted breast cancer cell proliferation via an epigenetic mechanism of FEN1-mediated up-regulation of DNA methyltransferase (DNMT)1 and DNMT3a. FEN1 was proved to interact with DNMT3a through proliferating cell nuclear antigen (PCNA) to suppress microRNA (miR)-200a-5p expression mediated by methylation. Furthermore, miR-200a-5p was identified to repress breast cancer cell proliferation by inhibiting the expression of its target genes, hepatocyte growth factor (MET), and epidermal growth factor receptor (EGFR). Overall, our data surprisingly demonstrate that FEN1 promotes breast cancer cell growth via the formation of FEN1/PCNA/DNMT3a complex to inhibit miR-200a expression by DNMT-mediated methylation and to recover the target genes expression of miR-200a, MET, and EGFR. The novel epigenetic mechanism of FEN1 on proliferation promotion provides a significant clue that FEN1 might serve as a predictive biomarker and therapeutic target for breast cancer. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphisms (SNPs) are emerging as important biomarkers for disease diagnosis, prognostics and disease pathogenesis. However, it is still challenging to make highly specific and sensitive SNPs detection to distinguish target DNA with single-base difference, so that flap endonuclease 1 (FEN 1) manipulated semi-DNA walker reaction and catalytic hairpin assembly (CHA)-FRET loop for ultrasensitive SNP detection has been proposed. Herein, FEN 1 was employed to specifically recognize and cut the 5′ flap of three-base overlapping structure formed by only hybridizing with mutant target (MT). Simultaneously, FEN 1 drove MT hybridization to autonomously walk along downstream probe (DP) and the cut sequence (CS) from semi-DNA walker reaction was collected by magnetic bead-based separation to avoid false-positive signal. Next, the cleaved CS triggered CHA and generated fluorescence resonance energy transfer (FRET) via Cy3 and Cy5. FEN 1 manipulated semi-DNA walker reaction to specifically recognize the target DNA and combined CHA-FRET loop to dually amplify signal. Thus, the SNP biosensor demonstrated superior performance for ultrasensitive detection of mutant KRAS gene with a low detection limit as low as 80 aM and was successfully applied to monitor the expression of mutant KRAS gene in human cancer cell lysates. This strategy provides an ultrasensitive way for the detection of biomolecules and reveals an effective avenue for diseases diagnosis. • A semi-DNA walker biosensing system is designed for SNP signal amplification. • FEN1 is used as a single base recognition element. • Local high concentrations of downstream probes drive cyclic cutting reactions. • The magnetic separation system avoids false positive signals. • Dual signal amplification strategy is used to improve the sensitivity of this biosensor. [ABSTRACT FROM AUTHOR]

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. 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. [Display omitted] • A photothermal and colorimetric dual modes biosensor was developed for FEN1 detection. • Naked eyes or common thermometer had been used readout. • The concentration of FEN1 can be detected in less than 1 h. • This biosensor had been used to evaluate the targets in serum samples. [ABSTRACT FROM AUTHOR]

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that is involved in the occurrence and development of various types of tumors. Previous studies have shown that FEN1 plays an important role in the development of hepatocellular carcinoma, however, the molecular mechanisms remain fully elucidated, especially its effect on the cell cycle of hepatocellular carcinoma has not been investigated. In this study, via bioinformatics prediction and clinical specimen verification, we confirmed that FEN1 was highly expressed in HCC and correlated with poor prognosis. The knockdown or overexpression of FEN1 could inhibit or promote the proliferation and invasion of HCC cells. Importantly, cell cycle and functional experiments showed that FEN1 could promote cell proliferation by inducing cell cycle transition from G2 to M phase. Further studies indicated that FEN1 regulated the G2/M transition by modulating cell division cycle 25C (Cdc25C), cyclin-dependent kinase 1 (CDK1) and Cyclin B1 expressions. To sum up, our research suggested that FEN1 could promote the proliferation, migration and invasion of HCC cells via activating cell cycle progression from G2 to M phase, indicating that FEN1 may be a potential target for the treatment of HCC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Human flap endonuclease 1 (FEN1) is a structure-specific, multi-functional endonuclease essential for DNA replication and repair. We and others have shown that during DNA replication, FEN1 processes Okazaki fragments via its interaction with the proliferating cell nuclear antigen (PCNA). Alternatively, 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 homology-directed repair of stalled DNA replication forks. However, it is unclear how FEN1 is able to switch between these interactions and its roles in DNA replication and DNA repair. Here, we report that FEN1 undergoes SUMOylation by SUMO-1 in response to DNA replication fork-stalling agents, such as UV irradiation, hydroxyurea, and mitomycin C. This DNA damage-induced SUMO-1 modification promotes the interaction of FEN1 with the Rad9–Rad1–Hus1 complex. Furthermore, we found that FEN1 mutations that prevent its SUMO-1 modification also impair its ability to interact with HUS1 and to rescue stalled replication forks. These impairments lead to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of the SUMO-1 modification of FEN1 in regulating its roles in DNA replication and repair. [ABSTRACT FROM AUTHOR]

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. 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. 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. [Display omitted] • FEN1 can be used as a cancer biomarkers. • Our method harnesses FEN1's enzymatic activity, enabling simpler & faster detection. [ABSTRACT FROM AUTHOR]

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. • We construct a single-molecule biosensor for FEN1 activity assay. • Dual signal amplification is introduced to achieve high sensitivity. • This biosensor can screen FEN1 inhibitors. • This biosensor can measure endogenous FEN1 activity at single-cell level. • This biosensor can detect FEN1 activity in breast cancer tissues. [ABSTRACT FROM AUTHOR]

Trastuzumab has been widely applied as a treatment for human epidermal growth factor 2 (HER2)- overexpressing breast cancer. However, the therapeutic efficacy of trastuzumab is limited. Flap endonuclease 1 (FEN1) is a multifunctional endonuclease that has a crucial role in DNA recombination and repair. Inhibition of FEN1 is associated with the reversal of anticancer drug resistance. However, it is unclear whether FEN1 is involved in trastuzumab resistance. In the present study, it was demonstrated that trastuzumab increases the expression of FEN1, and FEN1 knockdown significantly enhanced the sensitivity of BT474 cells to trastuzumab (P<0.05). It was also revealed that trastuzumab induced HER receptor activation, increased binding with FEN1 and estrogen receptor a (ERα), and upregulated ERα-target gene transcription (P<0.05). Upon silencing of FEN1 expression with siRNA, activation of HER receptor and FEN1 binding to ERα were decreased, and trastuzumab-induced ERα target gene upregulation was partially ameliorated (P<0.05). These results suggest that FEN1 may mediate trastuzumab resistance via inducing HER receptor activation and enhancing ERα-target gene transcription. The findings of the present study indicate a novel role of FEN1 in trastuzumab resistance, suggesting that targeting FEN1 may enhance the efficiency of trastuzumab as a treatment for HER2-positive breast cancer. [ABSTRACT FROM AUTHOR]

Lung cancer is one of the leading causes of cancer mortality worldwide. The therapeutic effect of chemotherapy is limited due to the resistance of cancer cells, which remains a challenge in cancer therapeutics. In this work, we found that flap endonuclease 1 ( FEN1) is overexpressed in lung cancer cells. FEN1 is a major component of the base excision repair pathway for DNA repair systems and plays important roles in maintaining genomic stability through DNA replication and repair. We showed that FEN1 is critical for the rapid proliferation of lung cancer cells. Suppression of FEN1 resulted in decreased DNA replication and accumulation of DNA damage, which subsequently induced apoptosis. Manipulating the amount of FEN1 altered the response of lung cancer cells to chemotherapeutic drugs. A small-molecule inhibitor (C20) was used to target FEN1 and this enhanced the therapeutic effect of cisplatin. The FEN1 inhibitor significantly suppressed cell proliferation and induced DNA damage in lung cancer cells. In mouse models, the FEN1 inhibitor sensitized lung cancer cells to a DNA damage-inducing agent and efficiently suppressed cancer progression in combination with cisplatin treatment. Our study suggests that targeting FEN1 may be a novel and efficient strategy for a tumor-targeting therapy for lung cancer. [ABSTRACT FROM AUTHOR]

Flap endonuclease 1 (FEN1) phosphorylation is proposed to regulate the action of FEN1 inDNArepair as well as Okazaki fragment maturation. However, the biologic significance of FEN1 phosphorylation in response to DNA damage remains unknown. Here, we report an in vivo role for FEN1 phosphorylation, using a mouse line carrying S187A FEN1, which abolishes FEN1 phosphorylation. Although S187A mouse embryonic fibroblast cells showed normal proliferation under low oxygen levels (2%), the mutant cells accumulated oxidative DNA damage, activated DNA damage checkpoints, and showed G1‐phase arrest at atmospheric oxygen levels (21%). This suggests an essential role for FEN1 phosphorylation in repairing oxygen‐induced DNA damage and maintaining proper cell cycle progression. Consistently, the mutant cardiomyocytes showed G1‐phase arrest due to activation of the p53‐mediated DNA damage response at the neonatal stage, which reduces the proliferation potential of the cardiomyocytes and impairs heart development. Nearly 50%of new borns with the S187 A mutant died in the first week due to failure to undergo the peroxisome proliferator‐activated receptor signaling‐dependent switch from glycolysis to fatty acid oxidation. The adult mutant mice developed dilated hearts and showed significantly shorter life spans. Altogether, our results reveal an important role of FEN1 phosphorylation to counteract oxygen‐induced stress in the heart during the fetal‐to‐neonatal transition.—Zhou, L., Dai, H., Wu, J., Zhou, M., Yuan, H., Du, J., Yang, L., Wu, X., Xu, H., Hua, Y., Xu, J., Zheng, L., Shen, B. Role of FEN1 S187 phosphorylation in counteracting oxygen‐induced stress and regulating postnatal heart development. FASEB J. 31, 132–147 (2017) www.fasebj.org [ABSTRACT FROM AUTHOR]

Flap endonuclease 1 (FEN1), a DNA repair protein, is important in preventing carcinogenesis. Two functional germ line variants -69G>A (rs174538) and +4150G>T (rs4246215) in the FEN1 gene have been associated with risk of various types of cancer. The aim of the present study was to evaluate the possible impact of FEN1 polymorphisms on risk of breast cancer (BC) in a sample of Iranian subjects. The FEN1 -69G>A and +4150G>T polymorphisms were analyzed in a case-control study that included 266 BC patients and 225 healthy females. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to geno-type the variants. The findings demonstrated that the FEN1 -69G>A and +4150G>T polymorphisms were not associated with BC risk in co-dominant, dominant and recessive inheritance models. The findings indicated that GG/GT, GA/GG and GA/TT genotypes significantly decreased the risk of BC when compared with -69GG/+4150GG. Furthermore, haplotype analysis indicated that -69G/+4150T as well as -69A/+4150G significantly decreased the risk of BC compared with -69G/+4150G. Thus, these findings demonstrated that haplotypes of FEN1 -69G>A and +4150G>T polymorphisms decreased the risk of BC in an Iranian population. Further studies with larger sample sizes and different ethnicities are required to validate the present findings. [ABSTRACT FROM AUTHOR]

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER – adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) – promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

DNA damage repair is one of the foremost factors leading to changes in tumor drug resistance. The analysis of Flap endonuclease 1 (FEN1), a kind of pivotal enzyme in various DNA metabolic pathways, has been of great support to tumor research and the development of chemotherapeutics. Nevertheless, few analytical techniques can achieve quantitative and simplified FEN1 measurement. Here, we constructed a double-wing switch nanodevice (DWSN)-mediated primer exchange technique for rapid and label-free quantification of FEN1 activity. Target FEN1 triggered the generation of numerous telomeric repeat fragments in different lengths through recognizing the three-base mismatched sites on the DWSN to release the 5′-Flaps. Further binding to the fluorescent dye ThT resulted in significantly enhanced fluorescence. This study broke the limitation of traditional single-site identification and demonstrated good sensitivity and specificity with detection limits up to 0.55 mU. Besides, the extraordinary analytical performance allowed the method to be utilized to monitor FEN1 extracted from cells and clinical serum samples and to compare the effect of targeted FEN1 inhibitors. • The introduction of DWSN provided an improved direction for the recognition efficiency. • This work broadened the applications of PER-based sensors to biological enzymes. • The sensor supported analysis of FEN1 in nuclear and cytoplasmic protein extracts. • Excellent S/B ratio gave this method leading analysis performance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Flap endonuclease 1 (FEN1), which is key in DNA replication and repair, has been demonstrated to be intimately involved in the development and progression of cancer. Our previous study determined that the downregulation of FEN1 can suppress the proliferation of, and induce apoptosis in, gastric cancer SGC-7901 cells. In addition, several FEN1 inhibitors have been identified to increase sensitisation to DNA injury agents. These results may provide a promising treatment method to enhance the traditional chemotherapeutics used for the treatment of gastric cancer. Thus, the aim of the present study was to determine the role of FEN1 in the chemosensitivity of SGC-7901 cells. The protein expression levels of FEN1 in cisplatin (CDDP)-treated SGC-7901 cells were detected using western blot analysis. FEN1 was silenced via specific FEN1-targeted small interfering RNAs (siRNA). The survival and apoptotic rates of the SGC-7901 cells were assessed using an MTT assay and flow cytometry, respectively. Relevant apoptotic factors were detected using western blotting. The results showed that the expression of FEN1 was significantly induced by CDDP in a dose- and time-dependent manner. The targeting of FEN1 in SGC-7901 cells, in combination with CDDP treatment, significantly inhibited their proliferation and effectively increased their apoptotic rate. In addition, in the cells targeted with FEN1-siRNA and exposed to CDDP, the levels of Bcl-2-associated X protein were significantly increased, whereas the expression levels of Bcl-2 and Bcl-extra large were effectively decreased, compared with the cells exposed to negative control-siRNA and CDDP. These results suggest a potential chemotherapeutic target, which exhibits enhanced sensitivity to CDDP following FEN1 silencing in SGC-7901 cells via decreased survival and increased apoptosis. [ABSTRACT FROM AUTHOR]

During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease ( FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing ( AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5′ ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is a neurological genetic disorder caused by the expansion of the CAG trinucleotide repeats (TNR) in the N-terminal region of coding sequence of the Huntingtin's (HTT) gene. This results in the addition of a poly-glutamine tract within the Huntingtin protein, resulting in its pathological form. The mechanism by which TRN expansion takes place is not yet fully understood. We have recently shown that DNA polymerase (Pol) β can promote the microhomology-mediated end joining and triplet expansion of a substrate mimicking a double strand break in the TNR region of the HTT gene. Here we show that TNR expansion is dependent on the structure of the DNA substrate, as well as on the two essential Pol β co-factors: flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). We found that Fen1 significantly stimulated TNR expansion by Pol β, but not by the related enzyme Pol λ, and subsequent ligation of the DNA products by Lig1. Interestingly, the deletion of N-terminal domains of Pol λ, resulted in an enzyme which displayed properties more similar to Pol β, suggesting a possible evolutionary mechanism. These results may suggest a novel mechanism for somatic TNR expansion in HD. [ABSTRACT FROM AUTHOR]

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 2 -MSNs) with a rich porous structure as the fluorescence nanoprobes to entrap the rhodamine 6G (Rh6G) molecules. Then gold nanoparticles linked specific single-stranded DNA (AuNPs-ssDNA) as a molecular gate was used to coat the NH 2 -MSNs surface. The fluorescence signal was weak when the fluorescence molecules were blocked by the AuNPs-ssDNA. In the presence of FEN1, it recognized and cleaved the specific ssDNA to release the Rh6G from NH 2 -MSNs, which resulted in recovered fluorescence signals. Thus, the sensitive detection of FEN1 activity was realized by controlled-release of Rh6G. The fluorescence signal showed a good linear relationship with the logarithm of FEN1 activity ranging from 0.05 to 1.75 U with a detection limit of 0.03 U. Moreover, confocal imaging demonstrated that the proposed biosensor could distinguish tumor cells from normal cells. Therefore, this technique contributes to clinical diagnostic and therapeutic monitoring. [ABSTRACT FROM AUTHOR]

An ultrasensitive homogeneous electrochemiluminescence biosensor for FEN1 detection had been developed on the basis of branched hybridization chain reaction (BHCR) amplification and ultrafiltration technology. [Display omitted] • An ultrasensitive ECL biosensor for FEN1 detection had been proposed. • Homogeneous strategy avoids tedious and cumbersome electrode modification. • Branched hybridization chain reaction was applied to increase the sensitivity. • The biosensor can be used to evaluate FEN1 activity in cells and serum samples. 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) 3 2+ can insert dsDNA to form Ru-dsDNAs, which can be easily separated from the main solution through ultrafiltration. The ECL signal from the separated Ru-dsDNAs has a good linear relationship with the logarithm of the FEN1 concentration ranging from 6.5 × 10−2 ∼ 6.5 × 103 U/L with a detection limit of 2.2 × 10−2 U/L. The proposed biosensor was used to evaluate FEN1 activity in real samples with satisfactory results. [ABSTRACT FROM AUTHOR]

Objective: Flap endonuclease 1 (FEN1) has been confirmed to involve the drug resistance of multiple cancers including breast cancer. However, the effect of miRNA-mediated FEN1 on breast cancer cell resistance is still ambiguous and needs further research., Methods: Firstly, we used GEPIA2 to predict the FEN1 expression in breast cancer. Next, we used quantitative real-time polymerase chain reaction (qRT-PCR) and western blot to evaluate the FEN1 level of cells. After parental cells or MDA-MB-231-paclitaxel (PTX) cells being transfected with or without siFEN1, the apoptosis, migration, and protein levels of FEN1, Bcl-2, and resistance-related genes were examined by flow cytometry, wound healing assay, and western blot, respectively. Then, the putative miRNA targeting FEN1 was predicted using StarBase V3.0, and further confirmed by qRT-PCR. The targeted binding of FEN1 to miR-26a-5p was detected by dual-luciferase reporter assay. After parental cells or MDA-MB-231-PTX cells being transfected with or without miR-26a-5p mimic, the apoptosis, migration, and protein levels of FEN1, Bcl-2, and resistance-related genes were tested again., Results: FEN1 expression was enhanced in breast cancer and MDA-MB-231-PTX cells. The combined application of FEN1 knockdown and PTX enhanced apoptosis in MDA-MB-231-PTX cells but suppressed cell migration and expressions of FEN1, Bcl-2, and resistance-related genes. Then, we confirmed that FEN1 was targeted by miR-26a-5p. The combined application of miR-26a-5p mimic and PTX largely facilitated apoptosis in MDA-MB-231-PTX cells but restrained cell migration and expressions of FEN1, Bcl-2, and resistance-related genes., Conclusion: MiR-26a-5p contributes to the sensitivity of breast cancer cells to paclitaxel via restraining FEN1., (© 2023 by the Association of Clinical Scientists, Inc.)

Oxidative stress is implicated in the pathogenesis of many diseases, including serious ocular diseases, keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). Flap endonuclease 1 (FEN1) plays an important role in the repair of oxidative DNA damage in the base excision repair pathway. We determined the association between two single nucleotide polymorphisms (SNPs), c.-441G>A (rs174538) and g.61564299G>T (rs4246215), in the FEN1 gene and the occurrence of KC and FECD. This study involved 279 patients with KC, 225 patients with FECD and 322 control individuals. Polymerase chain reaction (PCR) and length polymorphism restriction fragment analysis (RFLP) were applied. The T/T genotype of the g.61564299G>T polymorphism was associated with an increased occurrence of KC and FECD. There was no association between the c.-441G>A polymorphism and either disease. However, the GG haplotype of both polymorphisms was observed more frequently and the GT haplotype less frequently in the KC group than the control. The AG haplotype was associated with increased FECD occurrence. Our findings suggest that the g.61564299G>T and c.-441G>A polymorphisms in the FEN1 gene may modulate the risk of keratoconus and Fuchs endothelial corneal dystrophy. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphisms (SNPs) are main causes of differences in human phenotypes and drug resistance, especially the development of genetic diseases. Monitoring SNPs is of significant benefit to drug design and disease diagnosis. In this work, on the basis of tripartite DNAzyme junction formation induced by flap endonuclease 1 (FEN1) invader assay-triggered catalytic hairpin assembly (CHA), we describe an amplified and highly sensitive fluorescent strategy for detecting SNP of K-ras gene with substantial discrimination capability. The mutant DNA (MtDNA) of K-ras gene hybridizes with the sensing probe to inhibit the enzymatic activity of FEN1 to trigger subsequent CHA of three hairpins for the formation of tripartite DNAzyme junctions. And, the fluorescently quenched signal probes are efficiently and cyclically cleaved by the DNAzymes to restore largely magnified fluorescence for detecting MtDNA target sequence at 4.23 fM. Besides, the existence of low levels of MtDNA strands in diluted human serums and high concentrations of wild DNA solutions can be differentiated by such a method, showing its high potential for monitoring various SNPs for biological research and disease diagnosis. [Display omitted] • The sensitive and selective assay for SNP detection is developed. • Target-induced CHA formation of multi-DNAzyme junction leads to amplified signal. • Highly sensitive and selective discrimination of single nucleotide variation is realized. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two forms of human Pol δ were characterized in a reconstituted system. [•] Pol δ4 exhibits strong strand displacement activity in contrast to Pol δ3. [•] Primary cleavage products in the coupled Pol δ/Fen1 reactions were mononucleotides. [•] Pol δ3 exhibits near-ideal properties for a role in Okazaki fragment processing. [•] These studies support the novel concept that Pol δ3 participates in DNA replication. [Copyright &y& Elsevier]

Abstract: We examined a role for DNA polymerase β (Pol β) in mammalian long patch base excision repair (LP BER). Although a role for Pol β is well known in single-nucleotide BER, information on this enzyme in the context of LP BER has been limited. To examine the question of Pol β involvement in LP BER, we made use of nucleotide excision repair-deficient human XPA cells expressing UVDE (XPA-UVDE), which introduces a nick directly 5′ to the cyclobutane pyrimidine dimer or 6-4 photoproduct, leaving ends with 3′-OH and 5′-phosphorylated UV lesion. We observed recruitment of GFP-fused Pol β to focal sites of nuclear UV irradiation, consistent with a role of Pol β in repair of UV-induced photoproducts adjacent to a strand break. This was the first evidence of Pol β recruitment in LP BER in vivo. In cell extract, a 5′-blocked oligodeoxynucleotide substrate containing a nicked 5′-cyclobutane pyrimidine dimer was repaired by Pol β-dependent LP BER. We also demonstrated Pol β involvement in LP BER by making use of mouse cells that are double null for XPA and Pol β. These results were extended by experiments with oligodeoxynucleotide substrates and purified human Pol β. [Copyright &y& Elsevier]