Synergistic powering of DNA walker movement by endogenous dual enzymes for constructing dual-mode biosensors.

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.

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

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

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

One-Step Enzymatic Labeling Reveals a Critical Role of O-GlcNAcylation in Cell-Cycle Progression and DNA Damage Response.

O-linked N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous post-translational modification of proteins that is essential for cell function. Perturbation of O-GlcNAcylation leads to altered cell-cycle progression and DNA damage response. However, the underlying mechanisms are poorly understood. Here, we develop a highly sensitive one-step enzymatic strategy for capture and profiling O-GlcNAcylated proteins in cells. Using this strategy, we discover that flap endonuclease 1 (FEN1), an essential enzyme in DNA synthesis, is a novel substrate for O-GlcNAcylation. FEN1 O-GlcNAcylation is dynamically regulated during the cell cycle. O-GlcNAcylation at the serine 352 of FEN1 disrupts its interaction with Proliferating Cell Nuclear Antigen (PCNA) at the replication foci, and leads to altered cell cycle, defects in DNA replication, accumulation of DNA damage, and enhanced sensitivity to DNA damage agents. Thus, our study provides a sensitive method for profiling O-GlcNAcylated proteins, and reveals an unknown mechanism of O-GlcNAcylation in regulating cell cycle progression and DNA damage response.

DNA and RNA editing without sequence limitation using the flap endonuclease 1 guided by hairpin DNA probes.

Here, we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, designated the HpSGN system, for both DNA and RNA editing without sequence limitation. The compact size of the HpSGN system make it an ideal candidate for in vivo delivery applications. In vitro biochemical studies showed that the HpSGN system required less nuclease to cleave ssDNA substrates than the SGN system we reported previously by a factor of ∼40. Also, we proved that the HpSGN system can efficiently cleave different RNA targets in vitro. The HpSGN system cleaved genomic DNA at an efficiency of ∼40% and ∼20% in bacterial and human cells, respectively, and knocked down specific mRNAs in human cells at a level of ∼25%. Furthermore, the HpSGN system was sensitive to the single base mismatch at the position next to the hairpin both in vitro and in vivo. Collectively, this study demonstrated the potential of developing the HpSGN system as a small, effective, and specific editing tool for manipulating both DNA and RNA without sequence limitation.

R-loops promote trinucleotide repeat deletion through DNA base excision repair enzymatic activities.

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 ß (pol ß) 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 ß loop/hairpin bypass synthesis and the resolution of TNR R-loops through BER. Consequently, this resulted in fewer TNRs synthesized by pol ß 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.

Structure of the processive human Pol δ holoenzyme.

In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ's activity in replicating the human genome.

Photo-affinity pulling down of low-affinity binding proteins mediated by post-translational modifications.

Weak and transient protein-protein interactions (PPIs) mediated by the post-translational modifications (PTMs) play key roles in biological systems. However, technical challenges to investigate the PTM-mediated PPIs have impeded many research advances. In this work, we develop a photo-affinity pull-down assay method to pull-down low-affinity binding proteins, thus for the screen of PTM-mediated PPIs. In this method, the PTM-mediated non-covalent interactions can be converted to the covalent interactions by the photo-activated linkage, so as to freeze frame the low-affinity binding interactions. The fabricated photo-affinity magnetic beads (PAMBs) ensure high specificity and resolution to capture the interacted proteins. Besides, the introduction of PEG passivation layer on PAMB has significantly reduced the non-specific interaction as compared to the traditional pull-down assay. For proof-of-concept, by using this newly developed assay method, we have identified a set of proteins that can interact with a specific methylation site on Flap Endonuclease 1 (FEN1) protein. Less interfering proteins (decreased over 80%) and more proteins sub-classes are profiled as compared to the traditional biotin-avidin pull-down system. Therefore, this new pull-down method may provide a useful tool for the study of low-affinity PPIs, and contribute to the discovery of potential targets for renewed PTM-mediated interactions that is fundamentally needed in biomedical research.

Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO-POL enzyme.

Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5' exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3'-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain-especially the O helix-to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3' exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO-POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.

Gene cloning and characterization of Tk1281, a flap endonuclease 1 from Thermococcus kodakarensis.

Flap endonuclease is a structure-specific nuclease which cleaves 5'-flap of bifurcated DNA substrates. Genome sequence of Thermococcus kodakarensis harbors an open reading frame, Tk1281, exhibiting high homology with archaeal flap endonucleases 1. The corresponding gene was cloned and expressed in Escherichia coli, and the gene product was purified to apparent homogeneity. Tk1281 was a monomer of 38 kDa and catalyzed the cleavage of 5'-flap from double-stranded DNA substrate containing single-stranded DNA flap. The highest cleavage activity was observed at 80 °C and pH 7.5. Under optimal conditions, Tk1281 exhibited apparent Vmax and Km values of 278 nmol/min/mg and 37 µM, respectively, against a 54-nucleotide double-stranded substrate containing a single-stranded 5'-flap of 27 nucleotides. A unique feature of Tk1281 is its highest activation in the presence of Co2+ and no activation with Mn2+. To the best of our knowledge, this is the first cloning and characterization of a flap endonuclease from the genus Thermococcus.

Sequence-Specific Probe-Mediated Isothermal Amplification for the Single-Copy Sensitive Detection of Nucleic Acid.

There is currently the lack of a method for precisely monitoring the progress of isothermal amplification reactions by means of sequence-specific fluorescent probes like the TaqMan probe used in the PCR system. Here, we created a circular fluorescent probe-mediated isothermal amplification (CFPA) method. This novel method uses two circular fluorescent probes and Bst DNA polymerase to construct an overlapping structure that can be cut off by flap structure-specific endonuclease 1, separating the fluorescence and quenching groups on the probes. The results showed single-copy sensitivity, ultrahigh specificity, stability (C.V. < 0.1), and anti-interference ability in detecting nucleic acid samples. A clinical trial demonstrated the perfect effectiveness of this method in the diagnosis of rotavirus infection and consistency with the gold standard method used in the clinic ( p > 0.05). In summary, we present a new, reliable, and precise isothermal amplification approach for applications in biomedical research and the clinical accurate diagnosis of pathogen infections.

Initial state of DNA-Dye complex sets the stage for protein induced fluorescence modulation.

Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an increase in local viscosity due to the presence of the protein residues. This explanation, however, denies the opposite effect of fluorescence quenching. This work offers a perspective for understanding PIFE mechanism and reports the observation of a phenomenon that we name protein-induced fluorescence quenching (PIFQ), which exhibits an opposite effect to PIFE. A detailed characterization of these two fluorescence modulations reveals that the initial fluorescence state of the labeled mediator (DNA) determines whether this mediator-conjugated dye undergoes PIFE or PIFQ upon protein binding. This key role of the mediator DNA provides a protocol for the experimental design to obtain either PIFQ or PIFE, on-demand. This makes the arbitrary nature of the current experimental design obsolete, allowing for proper integration of both PIFE and PIFQ with existing bulk and single-molecule fluorescence techniques.

Sensitive detection of DNA from Chlamydia trachomatis by using flap endonuclease-assisted amplification and graphene oxide-based fluorescence signaling.

A simple and sensitive method is described for the determination of DNA. It relies on the use of (a) an invasive reaction that is catalyzed by flap endonuclease 1 (FEN 1), and (b) graphene oxide (GO)-based fluorescence signalling. The presence of target DNA mediates the formation of the invasive structure, and this induces FEN 1 to catalyze multiple cycles of cleavage reaction at the junction, thereby liberating numerous fluorophore-labeled flaps. The released flaps are intentionally designed too short to be adsorbed onto GO. Hence, intense green fluorescence whose maximum emission is observed at 520 nm after excitation at 480 nm is restored even in the presence of GO. The method can be applied to the determination of target DNA from Chlamydia trachomatis, one of the major pathogenic bacteria causing sexually transmitted diseases. The assay is sensitive and specific with the limit of detection of 6.7 pM, and was applied to reliable determination of Chlamydia trachomatis DNA in human serum. Graphical abstract Flap endonuclease 1 (FEN 1)-catalyzed invasive reaction and graphene oxide (GO)-based fluorescence signalling are integrated to develop a novel and sensitive target DNA detection method.

Flap Endonuclease 1 Mutations A159V and E160D Cause Genomic Instability by Slowing Reaction on Double-Flap Substrates.

Flap endonuclease 1 (FEN1) is a structure-selective nuclease best known for its roles in the penultimate steps of Okazaki fragment maturation, long-patch base excision repair and ribonucleotide excision repair. To better understand the role of FEN1 in genome maintenance in yeast and mammals, FEN1 active site mutations (A159V and E160D) have been used as tools to dissect its involvement in DNA metabolic pathways. However, discrepancies concerning the biochemistry and molecular etiology of genomic instability when FEN1 function is altered exist. Here, a detailed biochemical and biophysical characterization of mouse FEN1 and mutants is presented. Kinetic measurements showed that the active site mutants A159V and E160D reduce the rates of hydrolysis under multiple- and single-turnover conditions on all substrates. Consistent with their dominant negative effects in heterozygotes, neither mutation affects the adoption of the substrate duplex arms in the bent conformation on the enzyme surface, although decreases in substrate binding affinity are observed. The ability of the mutants to induce the requisite local DNA conformational change near the scissile phosphate is adversely affected, suggesting that the ability to place the scissile phosphate optimally in the active site causes the reduction in rates of phosphate diester hydrolysis. Further analysis suggests that the A159V mutation causes the chemistry of phosphate diester hydrolysis to become rate-limiting, whereas the wild-type and E160D proteins are likely rate-limited by a conformational change. On the basis of these results, the proposed roles of FEN1 in genome maintenance derived from studies involving these mutations are reassessed.

Structural basis of 5' flap recognition and protein-protein interactions of human flap endonuclease 1.

Human flap endonuclease 1 (hFEN1) is a structure-specific nuclease essential for DNA replication and repair processes. hFEN1 has 5' flap removal activity, as well as gap endonuclease activity that is critical for restarting stalled replication forks. Here, we report the crystal structures of wild-type and mutant hFEN1 proteins in complex with DNA substrates, followed by mutagenesis studies that provide mechanistic insight into the protein-protein interactions of hFEN1. We found that in an α-helix forming the helical gateway of hFEN1 recognizes the 5' flap prior to its threading into the active site for cleavage. We also found that the ß-pin region is rigidified into a short helix in R192F hFEN1-DNA structures, suppressing its gap endonuclease activity and cycle-dependent kinase interactions. Our findings suggest that a single mutation at the primary methylation site can alter the function of hFEN1 and provide insight into the role of the ß-pin region in hFEN1 protein interactions that are essential for DNA replication and repair.

rs4246215 is targeted by hsa-miR1236 to regulate FEN1 expression but is not associated with Fuchs' endothelial corneal dystrophy.

Fuchs' Endothelial Corneal Dystrophy (FECD) is a genetically complex disorder that affects individuals above 40 years of age; molecular pathogenesis of its associated genes is poorly understood. This study aims at assessing the association of flap endonuclease 1 (FEN1) polymorphisms, c.-69G>A (rs174538) and c.4150G>T (rs4246215) with FECD. Comet assay analysis reaffirmed that endogenous DNA damage was greater in FECD individuals. However, genetic analysis in 79 FECD patients and 234 unrelated control individuals prove that both the FEN1 polymorphisms, c.-69G>A (rs174538) and c.4150G>T (rs4246215), failed to show any genetic association with the FECD disease phenotype. In silico analysis and luciferase reporter assay identified 'G' allele of the 3'UTR located FEN1 polymorphism c.4150G>T as the target for binding of hsa-miR-1236-3p. This study indicates that although FEN1 polymorphisms, c.-69G>A (rs174538) and c.4150G>T (rs4246215) are not genetically associated with FECD, its transcript regulation reported in other diseases such as lung cancer which are genetically associated by rs4246215 could be mediated through miRNA, hsa-miR-1236-3p.

A conserved loop-wedge motif moderates reaction site search and recognition by FEN1.

DNA replication and repair frequently involve intermediate two-way junction structures with overhangs, or flaps, that must be promptly removed; a task performed by the essential enzyme flap endonuclease 1 (FEN1). We demonstrate a functional relationship between two intrinsically disordered regions of the FEN1 protein, which recognize opposing sides of the junction and order in response to the requisite substrate. Our results inform a model in which short-range translocation of FEN1 on DNA facilitates search for the annealed 3'-terminus of a primer strand, which is recognized by breaking the terminal base pair to generate a substrate with a single nucleotide 3'-flap. This recognition event allosterically signals hydrolytic removal of the 5'-flap through reaction in the opposing junction duplex, by controlling access of the scissile phosphate diester to the active site. The recognition process relies on a highly-conserved 'wedge' residue located on a mobile loop that orders to bind the newly-unpaired base. The unanticipated 'loop-wedge' mechanism exerts control over substrate selection, rate of reaction and reaction site precision, and shares features with other enzymes that recognize irregular DNA structures. These new findings reveal how FEN1 precisely couples 3'-flap verification to function.

Regional conformational flexibility couples substrate specificity and scissile phosphate diester selectivity in human flap endonuclease 1.

Human flap endonuclease-1 (hFEN1) catalyzes the divalent metal ion-dependent removal of single-stranded DNA protrusions known as flaps during DNA replication and repair. Substrate selectivity involves passage of the 5'-terminus/flap through the arch and recognition of a single nucleotide 3'-flap by the α2-α3 loop. Using NMR spectroscopy, we show that the solution conformation of free and DNA-bound hFEN1 are consistent with crystal structures; however, parts of the arch region and α2-α3 loop are disordered without substrate. Disorder within the arch explains how 5'-flaps can pass under it. NMR and single-molecule FRET data show a shift in the conformational ensemble in the arch and loop region upon addition of DNA. Furthermore, the addition of divalent metal ions to the active site of the hFEN1-DNA substrate complex demonstrates that active site changes are propagated via DNA-mediated allostery to regions key to substrate differentiation. The hFEN1-DNA complex also shows evidence of millisecond timescale motions in the arch region that may be required for DNA to enter the active site. Thus, hFEN1 regional conformational flexibility spanning a range of dynamic timescales is crucial to reach the catalytically relevant ensemble.

Crystal structure and mutational analysis of Mycobacterium smegmatis FenA highlight active site amino acids and three metal ions essential for flap endonuclease and 5' exonuclease activities.

Mycobacterium smegmatis FenA is a nucleic acid phosphodiesterase with flap endonuclease and 5' exonuclease activities. The 1.8 Å crystal structure of FenA reported here highlights as its closest homologs bacterial FEN-family enzymes ExoIX, the Pol1 exonuclease domain and phage T5 Fen. Mycobacterial FenA assimilates three active site manganese ions (M1, M2, M3) that are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. We find via mutagenesis that the carboxylate contacts to all three manganese ions are essential for FenA's activities. Structures of nuclease-dead FenA mutants D125N, D148N and D208N reveal how they fail to bind one of the three active site Mn2+ ions, in a distinctive fashion for each Asn change. The structure of FenA D208N with a phosphate anion engaged by M1 and M2 in a state mimetic of a product complex suggests a mechanism for metal-catalyzed phosphodiester hydrolysis similar to that proposed for human Exo1. A distinctive feature of FenA is that it does not have the helical arch module found in many other FEN/FEN-like enzymes. Instead, this segment of FenA adopts a unique structure comprising a short 310 helix and surface ß-loop that coordinates a fourth manganese ion (M4).

Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage.

Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair, and recombination and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps, from substrate binding to product release, during the hFEN1-catalyzed reaction in the presence of Mg2+ We found that whereas hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5'-flap:template junction, indicating that local DNA distortion (e.g. disruption of base stacking observed in structures), associated with positioning the 5'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34 and Asp-181), steering the 5'-flap through the active site and binding the scissile phosphate (Lys-93 and Arg-100), and stacking against the base 5' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5'-flaps.