DNA Processing Enzymes Research Articles

Enzymatic bypass of G-quadruplex structures containing oxidative lesions.

The function of many DNA processing enzymes involves sliding along the double helix or individual DNA strands. Stable secondary structures in the form of G-quadruplexes are difficult for such enzymes to bypass. We used a polymerase stop assay to determine which structural features of the human telomeric and the BCL2 promoter G-quadruplexes can stall progression of the Klenow fragment. Primer extension profiles revealed that G-quartets are effective roadblocks for the Klenow fragment, while auxiliary base pairs can be easily bypassed. Furthermore, we utilized 8-oxoguanine to simulate oxidative damage in G-rich regions and determine the effects on enzyme bypass. In rare cases, oxidative lesions reduce the level of G-quadruplex bypass. In general, however, oxidative lesions reduce G-quadruplex stability and facilitate bypassing of such G-rich regions, especially if the lesion persists in unfolding intermediates. Our findings using Klenow fragment can be extrapolated to other G-quadruplex forming sequences and enzymes that utilise a clamp-like structure to slide along DNA and are involved in processes such as gene expression regulation and telomere maintenance.

SNM1A is crucial for efficient repair of complex DNA breaks in human cells

DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-β-lactamase fold 5′−3′ exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.

Horizontal magnetic tweezers and its applications in single molecule micromanipulation experiments.

Magnetic tweezers (MTs) have become indispensable tools for gaining mechanistic insights into the behavior of DNA-processing enzymes and acquiring detailed, high-resolution data on the mechanical properties of DNA. Currently, MTs have two distinct designs: vertical and horizontal (or transverse) configurations. While the vertical design and its applications have been extensively documented, there is a noticeable gap in comprehensive information pertaining to the design details, experimental procedures, and types of studies conducted with horizontal MTs. This article aims to address this gap by providing a concise overview of the fundamental principles underlying transverse MTs. It will explore the multifaceted applications of this technique as an exceptional instrument for scrutinizing DNA and its interactions with DNA-binding proteins at the single-molecule level.

Chromatinization modulates topoisomerase II processivity

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress. While cellular processes constantly create varying torsional stress, how this variation impacts type IIA topoisomerase function remains obscure. Using multiple single-molecule approaches, we examined the torsional dependence of eukaryotic topoisomerase II (topo II) activity on naked DNA and chromatin. We observed that topo II is ~50-fold more processive on buckled DNA than previously estimated. We further discovered that topo II relaxes supercoiled DNA prior to plectoneme formation, but with processivity reduced by ~100-fold. This relaxation decreases with diminishing torsion, consistent with topo II capturing transient DNA loops. Topo II retains high processivity on buckled chromatin (~10,000 turns) and becomes highly processive even on chromatin under low torsional stress (~1000 turns), consistent with chromatin’s predisposition to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function.

Potential alteration enabled peptide elongation for direct serum detection of S100B in the early screening of phenylketonuria

DNA and aptamers have introduced new possibilities for utilizing synthetic probes in biosensing, effectively substituting antibodies. This substitution not only significantly cuts costs but also preserves analytical performance. However, in some cases, other biomacromolecules such as DNA processing enzymes remain indispensable for achieving signal amplification and high sensitivity. In this context, a novel bioassay has been developed, entirely eliminating the need for biomacromolecules. Instead, this approach employs fully synthesizable and modifiable peptides as the sensing probes. In lieu of enzymes, the strategy involves utilizing electrochemical potentials to guide peptide strand cleavage and the cross-coupling of peptide side chains at specific amino acids. This process mimics DNA-like chain elongation. The technique has already demonstrated success in detecting peripheral blood S100B levels, offering an early screening method for phenylketonuria in infants. These promising results suggest that the novel approach of utilizing electrochemical potentials to direct peptide bonding and cross-linking could find practical applications in clinical scenarios in the near future.

A real-time fluorescent gp32 probe-based assay for monitoring single-stranded DNA-dependent DNA processing enzymes

Single-stranded DNA (ssDNA) generated during DNA replication, recombination and damage repair reactions is an important intermediate and ssDNA-binding proteins that binds these intermediates coordinate various DNA metabolic processes. Mechanistic details of these ssDNA-dependent processes can be explored by monitoring the generation and consumption of ssDNA in real time. In this work, a fluorescein-labeled gp32-based sensor was employed to continuously monitor various aspects of ssDNA-dependent DNA replication and recombination processes in real time. The gp32 protein probe displayed high sensitivity and specificity to a variety of ssDNA-dependent processes of T4 phage. Several applications of the probe are illustrated here: the solution dynamics of ssDNA-binding protein, protein-protein and protein-DNA interactions involving gp32 protein and its mode of interaction, ssDNA translocation and protein displacement activities of helicases, primer extension activity of DNA polymerase holoenzyme and nucleoprotein filament formation during DNA recombination. The assay has identified new protein-protein interactions of gp32 during T4 replication and recombination. The fluorescent probe described here can thus be used as a universal probe for monitoring in real time various ssDNA-dependent processes, which is based on a well-characterized and easy-to-express bacteriophage T4 gene 32 protein, gp32.

DNA repair enzymes of the Antarctic Dry Valley metagenome.

Microbiota inhabiting the Dry Valleys of Antarctica are subjected to multiple stressors that can damage deoxyribonucleic acid (DNA) such as desiccation, high ultraviolet light (UV) and multiple freeze-thaw cycles. To identify novel or highly-divergent DNA-processing enzymes that may enable effective DNA repair, we have sequenced metagenomes from 30 sample-sites which are part of the most extensive Antarctic biodiversity survey undertaken to date. We then used these to construct wide-ranging sequence similarity networks from protein-coding sequences and identified candidate genes involved in specialized repair processes including unique nucleases as well as a diverse range of adenosine triphosphate (ATP) -dependent DNA ligases implicated in stationary-phase DNA repair processes. In one of the first direct investigations of enzyme function from these unique samples, we have heterologously expressed and assayed a number of these enzymes, providing insight into the mechanisms that may enable resident microbes to survive these threats to their genomic integrity.

Functional hierarchy of PCNA-interacting motifs in DNA processing enzymes.

Numerous eukaryotic DNA processing enzymes, such as DNA polymerases and ligases, bind the processivity factor PCNA, which acts as a platform to recruit and regulate the binding of enzymes to their DNA substrate. Multiple PCNA-interacting motifs (PIPs) are present in these enzymes, but their individual structural and functional role has been a matter of debate. Recent cryo-EM reconstructions of high-fidelity DNA polymerase Pol δ (Pol δ), translesion synthesis DNA polymerase κ (Pol κ) and Ligase 1 (Lig1) bound to a DNA substrate and PCNA demonstrate that the critical interaction with PCNA involves the internal PIP proximal to the catalytic domain. The ancillary PIPs, located in long disordered regions, are instead invisible in the reconstructions, and appear to function as flexible tethers when the enzymes fall off the DNA. In this review, we discuss the recent structural advancements and propose a functional hierarchy for the PIPs in Pol δ, Pol κ,and Lig1.

A single molecule TIRF-based platform to study DNA supercoiling effect on DNA processing enzymes.

DNA topology plays an important role in regulating DNA metabolic pathways. DNA supercoiling changes the DNA properties and proteins may differentially interact with DNA under various supercoiling states. A biophysical investigation of how proteins differentiate various topological states could further our understanding of protein-DNA interactions. We developed new single molecule total internal reflection fluorescence (smTIRF) -based platform to investigate how DNA topology affects protein-DNA interactions. We used this platform to study MutS, which detects a mismatched base pair to initiate mismatch repair, and CRISPR-Cas9. We generated plasmids that are site-specifically labeled with one or two fluorophores and a biotin. We prepared them in three different topological states: “Linear”, “relaxed circular”, and “negatively supercoiled”. For MutS study, we designed a Cy5 labeled plasmid with an adjacent mismatch. We observed FRET between Cy3-MutS and Cy5 when MutS bound the mismatch. In the presence of ADP, MutS bound the negative supercoiled DNA with a longer residence time compared to the relaxed circular DNA, suggesting that negative supercoiling helps mismatch recognition. For CRISPR-Cas9, we created a dual labeled plasmid to observe R-loop formation induced by dCas9. R-loop formation rate was similar for all three DNA topological states when the guide RNA sequence matched the target DNA sequence, while the rate was highest for the negatively supercoiled DNA and decreased progressively for the relaxed circular DNA and the linear DNA when we introduced one mismatch in either the PAM (protospacer adjacent motif)-proximal position or the PAM-distal position. Our observation confirms that DNA negative supercoiling assists DNA unwinding by Cas9, and this can exacerbate off-target effects in the cell. Collectively, our smTIRF-based platform allows high throughput investigation of supercoiling effects on DNA protein interactions as demonstrated using two different biological systems.

Probing single-molecule DNA-protein interactions using ABEL-FRET and ABEL-PIFE.

Single-molecule FRET and PIFE (protein induced fluorescence enhancement) are powerful techniques to probe molecular conformation and binding kinetics of DNA-protein interactions. We combine these techniques with Anti-Brownian Electrokinetic (ABEL) trapping to establish new modalities called ABEL-FRET and ABEL-PIFE. We demonstrate that ABEL-FRET achieves ultrahigh, shot-noise limited resolution of smFRET efficiency in solution without surface tethering. ABEL-PIFE enables simultaneous mapping of binding stoichiometry and sequence context of binding location. We highlight these new capabilities using examples of DNA processing enzymes.

A highly specific and flexible detection assay using collaborated actions of DNA-processing enzymes for identifying multiple gene expression signatures in breast cancer.

Most nucleic acid biosensors employ nucleic acid-processing enzymes to bind, degrade, splice, synthesize, and modify nucleic acids. Utilizing their unique substrate preference, binding mode, and catalytic activity is of great importance in designing nucleic acid biosensors. Combination with DNA-processing enzymes enables them to transform into a new generation of molecular diagnostics tools with enhanced selectivity and sensitivity and reduced reaction time. Here, we report an isothermal amplification strategy by coemploying a structure-specific endonuclease (flap endonuclease 1, FEN1) and a strand-displacing DNA polymerase (Bst DNA polymerase) to detect long RNA targets. This approach couples the FEN1-driven invasive cleavage reaction with toehold-mediated rolling circle amplification (iFEN-tRCA), enabling the highly selective and rapid detection of long RNA targets and offering a detection limit below 10 pM within 1 h. We used two targets, such as human epidermal growth factor receptor 2 (HER2, encoded by ERBB2) and dopamine- and cyclic AMP-regulated phosphoprotein (DARPP, encoded by PPP1R1B), associated with prognosis or response to anticancer therapy. We demonstrated the feasibility and quantitative capability of the iFEN-tRCA assay by assessing the expression of two RNA transcripts (ERBB2 and PPP1R1B) with total RNA extracts purified from human breast cancer cells. Therefore, we envision that the developed assay will provide a suitable prognostic and diagnostic tool for identifying appropriate patients for HER2-targeted therapy and predicting the clinical outcome and occurrence of metastasis relapse in breast cancer.

DNA fluctuations reveal the size and dynamics of topological domains.

DNA supercoiling is a key regulatory mechanism that orchestrates DNA readout, recombination, and genome maintenance. DNA-binding proteins often mediate these processes by bringing two distant DNA sites together, thereby inducing (transient) topological domains. In order to understand the dynamics and molecular architecture of protein-induced topological domains in DNA, quantitative and time-resolved approaches are required. Here, we present a methodology to determine the size and dynamics of topological domains in supercoiled DNA in real time and at the single-molecule level. Our approach is based on quantifying the extension fluctuations-in addition to the mean extension-of supercoiled DNA in magnetic tweezers (MT). Using a combination of high-speed MT experiments, Monte Carlo simulations, and analytical theory, we map out the dependence of DNA extension fluctuations as a function of supercoiling density and external force. We find that in the plectonemic regime, the extension variance increases linearly with increasing supercoiling density and show how this enables us to determine the formation and size of topological domains. In addition, we demonstrate how the transient (partial) dissociation of DNA-bridging proteins results in the dynamic sampling of different topological states, which allows us to deduce the torsional stiffness of the plectonemic state and the kinetics of protein-plectoneme interactions. We expect our results to further the understanding and optimization of magnetic tweezer measurements and to enable quantification of the dynamics and reaction pathways of DNA processing enzymes in the context of physiologically relevant forces and supercoiling densities.

Condensin-driven loop extrusion on supercoiled DNA.

Condensin, a structural maintenance of chromosomes (SMC) complex, has been shown to be a molecular motor protein that organizes chromosomes by extruding loops of DNA. In cells, such loop extrusion is challenged by many potential conflicts, for example, the torsional stresses that are generated by other DNA-processing enzymes. It has so far remained unclear how DNA supercoiling affects loop extrusion. Here, we use time-lapse single-molecule imaging to study condensin-driven DNA loop extrusion on supercoiled DNA. We find that condensin binding and DNA looping are stimulated by positively supercoiled DNA, and condensin preferentially binds near the tips of supercoiled plectonemes. Upon loop extrusion, condensin collects nearby plectonemes into a single supercoiled loop that is highly stable. Atomic force microscopy imaging shows that condensin generates supercoils in the presence of ATP. Our findings provide insight into the topology-regulated loading and formation of supercoiled loops by SMC complexes and clarify the interplay of loop extrusion and supercoiling.

A metal ion–dependent conformational switch modulates activity of the Plasmodium M17 aminopeptidase

The metal-dependent M17 aminopeptidases are conserved throughout all kingdoms of life. This large enzyme family is characterized by a conserved binuclear metal center and a distinctive homohexameric arrangement. Recently, we showed that hexamer formation in Plasmodium M17 aminopeptidases was controlled by the metal ion environment, although the functional necessity for hexamer formation is still unclear. To further understand the mechanistic role of the hexameric assembly, here we undertook an investigation of the structure and dynamics of the M17 aminopeptidase from Plasmodium falciparum, PfA-M17. We describe a novel structure of PfA-M17, which shows that the active sites of each trimer are linked by a dynamic loop, and loop movement is coupled with a drastic rearrangement of the binuclear metal center and substrate-binding pocket, rendering the protein inactive. Molecular dynamics simulations and biochemical analyses of PfA-M17 variants demonstrated that this rearrangement is inherent to PfA-M17, and that the transition between the active and inactive states is metal dependent and part of a dynamic regulatory mechanism. Key to the mechanism is a remodeling of the binuclear metal center, which occurs in response to a signal from the neighboring active site and serves to moderate the rate of proteolysis under different environmental conditions. In conclusion, this work identifies a precise mechanism by which oligomerization contributes to PfA-M17 function. Furthermore, it describes a novel role for metal cofactors in the regulation of enzymes, with implications for the wide range of metalloenzymes that operate via a two-metal ion catalytic center, including DNA processing enzymes and metalloproteases.

Telomerase holoenzymes

Telomerase is unusual genome replication machinery. The eukaryotic reverse transcriptase ribonucleoprotein (RNP) supplements telomeric simple‐sequence repeat tracts at chromosome ends by new repeat synthesis. Using each chromosome 3’ end as primer and an internal region of the integral telomerase RNA subunit as template, telomerase reiteratively reverse transcribes its short template into tandem single‐stranded telomeric repeats. Lagging‐strand DNA replication machinery and processing enzymes then create the functional telomere structure of duplex repeats with a 3’ overhang that can be single‐stranded, form alternative DNA structures such as G‐quadruplex, or invade the duplex region to form a displacment loop (D‐loop) of telomeric repeats termed the t‐loop.Two and a half decades of research in the Collins lab led the way to a resolution for several puzzles that were facing the telomere and telomerase field since soon after telomerase discovery by Carol Greider and Elizabeth Blackburn. How do telomerase reverse transcriptase protein (TERT) and telomerase RNA co‐fold in a hierarchical, complex process of cellular telomerase RNP biogenesis, and how does this drastically limit the amount of telomerase made by even the most strongly telomerase‐positive human cancer and stem cells? How can a precisely defined region within telomerase RNA be used as template? How does the template‐product duplex unpair and reform in a different template register to enable processive repeat synthesis? How is telomerase brought to telomeres and how does each telomere control telomerase activity according to its individual length to generate telomere length homeostasis?Human telomerase RNA (hTR) relies on proteins shared with H/ACA‐family small nucleolar RNAs for its processing, stability, and assembly with TERT. Inherited and spontaneous mutations in these H/ACA proteins cause disease syndromes by reducing telomerase function, as do subsequently identified mutations in hTR and TERT. Understanding of how these mutations compromise telomerase function has emerged from new high‐resolution structures of telomerase holoenzymes from the ciliate Tetrahymena thermophila and human cells, the two model systems of Collins lab research, and from understanding control of telomerase function at telomeres.

Magnetic tweezers: development and use in single-molecule research.

The use of magnetic tweezers for single-molecule micromanipulation has evolved rapidly since its introduction approximately30years ago. Magnetic tweezers have provided important insights into the dynamic activity of DNA-processing enzymes, as well as detailed, high-resolution information on the mechanical properties of DNA. These successes have been enabled by major advancements in the hardware and software components of these devices. These developments now allow for a much richer mechanistic understanding of the functions and mechanisms of DNA-binding enzymes. In this review, the authors briefly discuss the fundamental principles of magnetic tweezers anddescribe the advancements that have made it a superlative tool for investigating, at the single-molecule level, DNA and its interactions with DNA-binding proteins.

A simple and general approach to generate photoactivatable DNA processing enzymes.

DNA processing enzymes, such as DNA polymerases and endonucleases, have found many applications in biotechnology, molecular diagnostics, and synthetic biology, among others. The development of enzymes with controllable activity, such as hot-start or light-activatable versions, has boosted their applications and improved the sensitivity and specificity of the existing ones. However, current approaches to produce controllable enzymes are experimentally demanding to develop and case-specific. Here, we introduce a simple and general method to design light-start DNA processing enzymes. In order to prove its versatility, we applied our method to three DNA polymerases commonly used in biotechnology, including the Phi29 (mesophilic), Taq, and Pfu polymerases, and one restriction enzyme. Light-start enzymes showed suppressed polymerase, exonuclease, and endonuclease activity until they were re-activated by an UV pulse. Finally, we applied our enzymes to common molecular biology assays and showed comparable performance to commercial hot-start enzymes.

Kinetics of γH2AX and phospho-histone H3 following pulse treatment of TK6 cells provides insights into clastogenic activity

The desire for in vitro genotoxicity assays to provide higher information content, especially regarding chemicals' predominant genotoxic mode of action, has led to the development of a novel multiplexed assay available under the trade name MultiFlow®. We report here on an experimental design variation that provides further insight into clastogens' genotoxic activity. First, the standard MultiFlow DNA Damage Assay-p53, γ H2AX, phospho-histone H3 was used with human TK6 lymphoblastoid cells that were exposed for 24 continuous hours to each of 50 reference clastogens. This initial analysis correctly identified 48/50 compounds as clastogenic. These 48 compounds were then evaluated using a short-term, 'pulse' treatment protocol whereby cells were exposed to test chemical for 4 h, a centrifugation/washout step was performed, and cells were allowed to recover for 20 h. MultiFlow analyses were accomplished at 4 and 24 h. The γ H2AX and phospho-histone H3 biomarkers were found to exhibit distinct differences in terms of their persistence across chemical classes. Unsupervised hierarchical clustering analysis identified three groups. Examination of the compounds within these groups showed one cluster primarily consisting of alkylators that directly target DNA. The other two groups were dominated by non-DNA alkylators and included anti-metabolites, oxidative stress inducers and chemicals that inhibit DNA-processing enzymes. These results are encouraging, as they suggest that a simple follow-up test for in vitro clastogens provides mechanistic insights into their genotoxic activity. This type of information will contribute to improve decision-making and help guide further testing.

An Autoinhibitory Role for the GRF Zinc Finger Domain of DNA Glycosylase NEIL3

DNA damage within a cell can cause damaging effects on genome stability if not properly corrected. Exogenous factors such as UV-light and environmental toxins, and endogenous factors including reactive metabolites and oxidative stress cause DNA damage in the form of modified DNA nucleobases. Base excision repair (BER) is the primary pathway to fix these lesions and begins with recognition and excision of the damaged nucleobase by DNA glycosylase enzymes. The DNA glycosylase, endonuclease VIII-like 3 (NEIL3), removes oxidized bases from single-stranded (ss) DNA, and unhooks interstrand cross-links (ICLs), which covalently tether opposing DNA strands and block transcription and replication. NEIL3 contains two C-terminal GRF-type zinc finger (Zf-GRFs) motifs absent in other NEIL paralogs and the role of the Zf-GRFs are unknown. Here we show the first crystal structure of the C-terminus of NEIL3 which includes a flexible head-to-tail configuration, predicted to bind ssDNA in many conformations. We show via electrophoretic mobility shift assays (EMSA) that the Zf-GRF domain binds with higher affinity than individual Zf-GRF motifs. Binding assays performed by fluorescence anisotropy showed DNA length dependence and DNA binding stoichiometry of the Zf-GRF domain. Functionally we show that the Zf-GRFs inhibit base excision activity of the NEIL3 glycosylase domain (NEIL3-GD) in trans and in cis. This autoinhibitory activity contrasts GRF-Zf domains of other DNA processing enzymes, which typically bind ssDNA to enhance catalytic activity, and suggests that the C-terminal of NEIL3 is involved in DNA damage recruitment and enzymatic regulation.

An autoinhibitory role for the GRF zinc finger domain of DNA glycosylase NEIL3

The NEIL3 DNA glycosylase maintains genome integrity during replication by excising oxidized bases from single-stranded DNA (ssDNA) and unhooking interstrand cross-links (ICLs) at fork structures. In addition to its N-terminal catalytic glycosylase domain, NEIL3 contains two tandem C-terminal GRF-type zinc fingers that are absent in the other NEIL paralogs. ssDNA binding by the GRF-ZF motifs helps recruit NEIL3 to replication forks converged at an ICL, but the nature of DNA binding and the effect of the GRF-ZF domain on catalysis of base excision and ICL unhooking is unknown. Here, we show that the tandem GRF-ZFs of NEIL3 provide affinity and specificity for DNA that is greater than each individual motif alone. The crystal structure of the GRF domain shows that the tandem ZF motifs adopt a flexible head-to-tail configuration well-suited for binding to multiple ssDNA conformations. Functionally, we establish that the NEIL3 GRF domain inhibits glycosylase activity against monoadducts and ICLs. This autoinhibitory activity contrasts GRF-ZF domains of other DNA-processing enzymes, which typically use ssDNA binding to enhance catalytic activity, and suggests that the C-terminal region of NEIL3 is involved in both DNA damage recruitment and enzymatic regulation.