Linear Duplex DNA Research Articles

Structural basis of DNA crossover capture by Escherichia coli DNA gyrase.

DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules, enabling the transport of one duplex through the transient double-stranded break of the other, remains elusive owing to structures derived solely from single linear duplex DNAs lacking topological constraints. Using cryo-electron microscopy, we solved the structure of Escherichia coli DNA gyrase bound to a negatively supercoiled minicircle DNA. We show how DNA gyrase captures a DNA crossover, revealing both conserved molecular grooves that accommodate the DNA helices. Together with molecular tweezer experiments, the structure shows that the DNA crossover is of positive chirality, reconciling the binding step of gyrase-mediated DNA relaxation and supercoiling in a single structure.

High spontaneous integration rates of end-modified linear DNAs upon mammalian cell transfection

In gene therapy, potential integration of therapeutic transgene into host cell genomes is a serious risk that can lead to insertional mutagenesis and tumorigenesis. Viral vectors are often used as the gene delivery vehicle, but they are prone to undergoing integration events. More recently, non-viral delivery of linear DNAs having modified geometry such as closed-end linear duplex DNA (CELiD) have shown promise as an alternative, due to prolonged transgene expression and less cytotoxicity. However, whether modified-end linear DNAs can also provide a safe, non-integrating gene transfer remains unanswered. Herein, we compare the genomic integration frequency upon transfection of cells with expression vectors in the forms of circular plasmid, unmodified linear DNA, CELiDs with thioester loops, and Streptavidin-conjugated blocked-end linear DNA. All of the forms of linear DNA resulted in a high fraction of the cells being stably transfected—between 10 and 20% of the initially transfected cells. These results indicate that blocking the ends of linear DNA is insufficient to prevent integration.

Reinforcing DNA Supramolecular Hydrogel with Polymeric Multiple-Unit Linker

Reinforcing DNA Supramolecular Hydrogel with Polymeric Multiple-Unit Linker

The mismatched nucleotides encoded in vaccinia virus flip-and-flop hairpin telomeres serve an essential role in virion maturation.

Poxvirus genomes consist of a linear duplex DNA that ends in short inverted and complementary hairpin structures. These elements also encode loops and mismatches that likely serve a role in genome packaging and perhaps replication. We constructed mutant vaccinia viruses (VACV) where the native hairpins were replaced by altered forms and tested effects on replication, assembly, and virulence. Our studies showed that structure, not sequence, likely determines function as one can replace an Orthopoxvirus (VACV) hairpin with one copied from a Leporipoxvirus with no effect on growth. Some loops can be deleted from VACV hairpins with little effect, but VACV bearing too few mismatches grew poorly and we couldn’t recover viruses lacking all mismatches. Further studies were conducted using a mutant bearing only one of six mismatches found in wild-type hairpins (SΔ1Δ3–6). This virus grew to ~20-fold lower titers, but neither DNA synthesis nor telomere resolution was affected. However, the mutant exhibited a particle-to-PFU ratio 10-20-fold higher than wild-type viruses and p4b/4b core protein processing was compromised, indicating an assembly defect. Electron microscopy showed that SΔ1Δ3–6 mutant development was blocked at the immature virus (IV) stage, which phenocopies known effects of I1L mutants. Competitive DNA binding assays showed that recombinant I1 protein had less affinity for the SΔ1Δ3–6 hairpin than the wild-type hairpin. The SΔ1Δ3–6 mutant was also attenuated when administered to SCID-NCR mice by tail scarification. Mice inoculated with viruses bearing wild-type hairpins exhibited a median survival of 30–37 days, while mice infected with SΔ1Δ3–6 virus survived >70 days. Persistent infections favor genetic reversion and genome sequencing detected one example where a small duplication near the hairpin tip likely created a new loop. These observations show that mismatches serve a critical role in genome packaging and provide new insights into how VACV “flip and flop” telomeres are arranged.

AAVS1 site-specific integration of the CAR gene into human primary T cells using a linear closed-ended AAV-based DNA vector.

Use of chimeric antigen receptor (CAR) T cells has become a promising strategy in cancer immunotherapy. However, safety in clinical application is also one of the most controversial issues. In the present study, we investigated the application of a non-viral site-directed vector (CELiD [closed-ended linear duplex DNA]) dependent on adeno-associated virus (AAV) genomes for the purpose of safe CAR-T engineering. We co-electroporated CD19-CAR encoding "CELiD" vectors with plasmid pCMV-Rep into human T cells and ensured stably transfected CAR-T cells by G418 selection. The efficiency of AAVS1 site-specific integration was analyzed by a real-time polymerase chain reaction. CAR-T cells engineered by CELiD vectors could be established within 20 days with up to 22.8% AAVS1 site-specific integration efficiency. CAR expression and cytokine secretion of CAR modified T cells were evaluated in vitro. Abundant effector cytokines were produced by the CAR-T cells engineered by CELiD vectors compared to control T cells and the killing efficiency of target cells was estimated to as high as 75% in vitro. With the help of the AAV-derived CELiD vector, CAR genes were preferentially integrated into the AAVS1 site. This technology could be utilized in human T cell modification and remove the safety constraints of CAR-T therapy.

BIOPHYSICS MEETS GENE THERAPY: HOW EXPLORING SUPERCOILING-DEPENDENT STRUCTURAL CHANGES IN DNA LED TO THE DEVELOPMENT OF MINIVECTOR DNA.

Supercoiling affects every aspect of DNA function (replication, transcription, repair, recombination, etc.), yet the vast majority of studies on DNA and crystal structures of the molecule utilize short linear duplex DNA, which cannot be supercoiled. To study how supercoiling drives DNA biology, we developed and patented methods to make milligram quantities of tiny supercoiled circles of DNA called minicircles. We used a collaborative and multidisciplinary approach, including computational simulations (both atomistic and coarse-grained), biochemical experimentation, and biophysical methods to study these minicircles. By determining the three-dimensional conformations of individual supercoiled DNA minicircles, we revealed the structural diversity of supercoiled DNA and its highly dynamic nature. We uncovered profound structural changes, including sequence-specific base-flipping (where the DNA base flips out into the solvent), bending, and denaturing in negatively supercoiled minicircles. Counterintuitively, exposed DNA bases emerged in the positively supercoiled minicircles, which may result from inside-out DNA (Pauling-like, or "P-DNA"). These structural changes strongly influence how enzymes interact with or act on DNA. We hypothesized that, because of their small size and lack of bacterial sequences, these small supercoiled DNA circles may be efficient at delivering DNA into cells for gene therapy applications. "Minivectors," as we named them for this application, have proven to have therapeutic potential. We discovered that minivectors efficiently transfect a wide range of cell types, including many clinically important cell lines that are refractory to transfection with conventional plasmid vectors. Minivectors can be aerosolized for delivery to lungs and transfect human cells in culture to express RNA or genes. Importantly, minivectors demonstrate no obvious vector-associated toxicity. Minivectors can be repeatedly delivered and are long-lasting without integrating into the genome. Requests from colleagues around the world for minicircle and minivector DNA revealed a demand for our invention. We successfully obtained start-up funding for Twister Biotech, Inc. to help fulfill this demand, providing DNA for those who needed it, with a long-term goal of developing human therapeutics. In summary, what started as a tool for studying DNA structure has taken us in new and unanticipated directions.

Transmission of dynamic supercoiling in linear and multi-way branched DNAs and its regulation revealed by a fluorescent G-quadruplex torsion sensor.

DNA supercoiling is an important regulator of gene activity. The transmission of transcription-generated supercoiling wave along a DNA helix provides a way for a gene being transcribed to communicate with and regulate its neighboring genes. Currently, the dynamic behavior of supercoiling transmission remains unclear owing to the lack of a suitable tool for detecting the dynamics of supercoiling transmission. In this work, we established a torsion sensor that quantitatively monitors supercoiling transmission in real time in DNA. Using this sensor, we studied the transmission of transcriptionally generated negative supercoiling in linear and multi-way DNA duplexes. We found that transcription-generated dynamic supercoiling not only transmits along linear DNA duplex but also equally diverges at and proceeds through multi-way DNA junctions. We also show that such a process is regulated by DNA–protein interactions and non-canonical DNA structures in the path of supercoiling transmission. These results imply a transcription-coupled mechanism of dynamic supercoiling-mediated intra- and inter-chromosomal signal transduction pathway and their regulation in DNA.

Reconstituting the 4-Strand DNA Strand Exchange.

Proteins of the Rad51 family play a key role in homologous recombination by carrying out DNA strand exchange. Here, we present the methodology and the protocols for the 4-strand exchange between gapped circular DNA and homologous linear duplex DNA promoted by human Rad51 and Escherichia coli RecA orthologs. This reaction includes formation of joint molecules and their extension by branch migration in a polar manner. The presented methodology may be used for reconstitution of the medial-to-late stages of homologous recombination in vitro as well as for investigation of the mechanisms of branch migration by helicase-like proteins, e.g., Rad54, BLM, or RecQ1.

Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity.

Much is known about the Escherichia coli nucleotide excision repair (NER) pathway; however, very little is understood about the proteins involved and the molecular mechanism of NER in mycobacteria. In this study, we show that Mycobacterium tuberculosis UvrB (MtUvrB), which exists in solution as a monomer, binds to DNA in a structure-dependent manner. A systematic examination of MtUvrB substrate specificity reveals that it associates preferentially with single-stranded DNA, duplexes with 3' or 5' overhangs, and linear duplex DNA with splayed arms. Whereas E. coli UvrB (EcUvrB) binds weakly to undamaged DNA and has no ATPase activity, MtUvrB possesses intrinsic ATPase activity that is greatly stimulated by both single- and double-stranded DNA. Strikingly, we found that MtUvrB, but not EcUvrB, possesses the DNA unwinding activity characteristic of an ATP-dependent DNA helicase. The helicase activity of MtUvrB proceeds in the 3' to 5' direction and is strongly modulated by a nontranslocating 5' single-stranded tail, indicating that in addition to the translocating strand it also interacts with the 5' end of the substrate. The fraction of DNA unwound by MtUvrB decreases significantly as the length of the duplex increases: it fails to unwind duplexes longer than 70 bp. These results, on one hand, reveal significant mechanistic differences between MtUvrB and EcUvrB and, on the other, support an alternative role for UvrB in the processing of key DNA replication intermediates. Altogether, our findings provide insights into the catalytic functions of UvrB and lay the foundation for further understanding of the NER pathway in M. tuberculosis.

Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli.

ABSTRACTRecombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion.

Serum viral duplex-linear DNA proportion increases with the progression of liver disease in patients infected with HBV

ObjectiveHBV has two forms of genomic DNA, relaxed-circular DNA (rcDNA) and duplex-linear DNA (dlDNA). Compared to rcDNA, dlDNA has been demonstrated to integrate more frequently into host cellular chromosomes, which...

Characterization of IHF Binding to DNA Four-Way Junctions and Forks

Integration Host Factor (IHF) is an architectural protein that binds and bends DNA, facilitating the formation of protein-DNA complexes important for gene regulation. IHF binds with high affinity to a specific consensus sequence and induces a 160° bend upon binding. We have shown that IHF binds DNA four way junctions (4WJ) that do not contain the consensus sequence with nanomolar affinity and 1:1 stoichiometry for the specific interaction. We have also observed that IHF binds DNA forks with nanomolar affinity. The binding to junctions and forks is in direct contrast to IHF binding to linear duplex DNA, which is typically 1000-fold weaker. In this study we investigate whether the presence of the IHF consensus sequence influences IHF binding to DNA junctions and forks. We utilized gel shift and fluorescence binding assays to measure affinity and have observed that the high affinity for these non-native structures is independent of the presence of the consensus sequence. We are further exploring how IHF binding influences junction conformation. Junction conformation is modulated by ion concentration where high concentrations of ions induces pairwise stacking of the helical arms resulting in quasi-continuous helices. In our investigations IHF binding to the non-consensus junction induces an open conformation. We are specifically examining the distortion of the junction and DNA fork substrates upon IHF binding using Forster Resonance Energy Transfer. Through these measurements, we are also exploring whether the mechanism of recognition differs between junctions and forks.

Internucleosomal DNA fragmentation in wild emmer wheat is catalyzed by S1-type endonucleases translocated to the nucleus upon induction of cell death

Leaves of cereal plants display nucleosomal fragmentation of DNA attributed to the action of nucleases induced during program cell death (PCD). Yet, the specific nuclease activity responsible for generating double strand DNA breaks (DSBs) that lead to DNA fragmentation has not been fully described. Here, we characterized a Ca2+/Mg2+-dependent S1-type endonuclease activity in leaves of wild emmer wheat (Triticum dicoccoides Köern.) capable of introducing DSBs as demonstrated by the conversion of supercoiled plasmid DNA into a linear duplex DNA. In-gel nuclease assay revealed a nuclease of about 35kDa capable of degrading both single stranded DNA and RNA. We further showed that the endonuclease activity can be purified on Concanavalin A and treatment with peptide-N-glycosidase F (PNGase F) did not abolish its activity. Furthermore, ConA-associated endonuclease was capable of generating nucleosomal DNA fragmentation in tobacco nuclei. Since S1-type endonucleases lack canonical nuclear localization signal it was necessary to determine their subcellular localization. To this end, a cDNA encoding for a putative 34kDa S1-type nuclease, designated TaS1-like (TaS1L) was synthesized based on available sequence data of Triticum aestivum and fused with RFP. Introduction into protoplasts showed that TaS1L-RFP is cytoplasmic 24h post transformation but gradually turn nuclear at 48h concomitantly with induction of cell death. Our results suggest that DNA fragmentation occurring in leaves of wild emmer wheat may be attributed to S1-type endonuclease(s) that reside in the cytoplasm but translocate to the nucleus upon induction of cell death.

Mycobacterium tuberculosis DinG Is a Structure-specific Helicase That Unwinds G4 DNA: Implications for Targeting G4 DNA as a Novel Therapeutic Approach

The significance of G-quadruplexes and the helicases that resolve G4 structures in prokaryotes is poorly understood. The Mycobacterium tuberculosis genome is GC-rich and contains >10,000 sequences that have the potential to form G4 structures. In Escherichia coli, RecQ helicase unwinds G4 structures. However, RecQ is absent in M. tuberculosis, and the helicase that participates in G4 resolution in M. tuberculosis is obscure. Here, we show that M. tuberculosis DinG (MtDinG) exhibits high affinity for ssDNA and ssDNA translocation with a 5' → 3' polarity. Interestingly, MtDinG unwinds overhangs, flap structures, and forked duplexes but fails to unwind linear duplex DNA. Our data with DNase I footprinting provide mechanistic insights and suggest that MtDinG is a 5' → 3' polarity helicase. Notably, in contrast to E. coli DinG, MtDinG catalyzes unwinding of replication fork and Holliday junction structures. Strikingly, we find that MtDinG resolves intermolecular G4 structures. These data suggest that MtDinG is a multifunctional structure-specific helicase that unwinds model structures of DNA replication, repair, and recombination as well as G4 structures. We finally demonstrate that promoter sequences of M. tuberculosis PE_PGRS2, mce1R, and moeB1 genes contain G4 structures, implying that G4 structures may regulate gene expression in M. tuberculosis. We discuss these data and implicate targeting G4 structures and DinG helicase in M. tuberculosis could be a novel therapeutic strategy for culminating the infection with this pathogen.

Simulations of DNA topoisomerase 1B bound to supercoiled DNA reveal changes in the flexibility pattern of the enzyme and a secondary protein-DNA binding site.

Human topoisomerase 1B has been simulated covalently bound to a negatively supercoiled DNA minicircle, and its behavior compared to the enzyme bound to a simple linear DNA duplex. The presence of the more realistic supercoiled substrate facilitates the formation of larger number of protein–DNA interactions when compared to a simple linear duplex fragment. The number of protein–DNA hydrogen bonds doubles in proximity to the active site, affecting all of the residues in the catalytic pentad. The clamp over the DNA, characterized by the salt bridge between Lys369 and Glu497, undergoes reduced fluctuations when bound to the supercoiled minicircle. The linker domain of the enzyme, which is implicated in the controlled relaxation of superhelical stress, also displays an increased number of contacts with the minicircle compared to linear DNA. Finally, the more complex topology of the supercoiled DNA minicircle gives rise to a secondary DNA binding site involving four residues located on subdomain III. The simulation trajectories reveal significant changes in the interactions between the enzyme and the DNA for the more complex DNA topology, which are consistent with the experimental observation that the protein has a preference for binding to supercoiled DNA.

Mismatch repair protein hMSH2-hMSH6 recognizes mismatches and forms sliding clamps within a D-loop recombination intermediate.

High fidelity homologous DNA recombination depends on mismatch repair (MMR), which antagonizes recombination between divergent sequences by rejecting heteroduplex DNA containing excessive nucleotide mismatches. The hMSH2-hMSH6 heterodimer is the first responder in postreplicative MMR and also plays a prominent role in heteroduplex rejection. Whether a similar molecular mechanism underlies its function in these two processes remains enigmatic. We have determined that hMSH2-hMSH6 efficiently recognizes mismatches within a D-loop recombination initiation intermediate. Mismatch recognition by hMSH2-hMSH6 is not abrogated by human replication protein A (HsRPA) bound to the displaced single-stranded DNA (ssDNA) or by HsRAD51. In addition, ATP-bound hMSH2-hMSH6 sliding clamps that are essential for downstream MMR processes are formed and constrained within the heteroduplex region of the D-loop. Moreover, the hMSH2-hMSH6 sliding clamps are stabilized on the D-loop by HsRPA bound to the displaced ssDNA. Our findings reveal similarities and differences in hMSH2-hMSH6 mismatch recognition and sliding-clamp formation between a D-loop recombination intermediate and linear duplex DNA.

DNA structure-specific priming of ATR activation by DNA-PKcs

Three phosphatidylinositol-3-kinase-related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)-covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs.

Real-time solution measurement of RAD51- and RecA-mediated strand assimilation without background annealing

RAD51 is the central strand exchange recombinase in somatic homologous recombination, providing genomic stability and promoting resistance to DNA damage. An important tool for mechanistic studies of RAD51 is the D-loop or strand assimilation assay, which measures the ability of RAD51-coated single-stranded DNA (ssDNA) to search for, invade and exchange ssDNA strands with a homologous duplex DNA target. As cancer cells generally overexpress RAD51, the D-loop assay has also emerged as an important tool in oncologic drug design programs for targeting RAD51. Previous studies have adapted the traditional gel-based D-loop assay by using fluorescence-based substrates, which in principle allow for use in high-throughput screening platforms. However, these existing D-loop methods depend on linear oligonucleotide DNA duplex targets, and these substrates enable recombinase-independent ssDNA annealing that can obscure the recombinase-dependent strand assimilation signal. This compelled us to fundamentally re-design this assay, using a fluorescent target substrate that consists of a covalently closed linear double-hairpin dsDNA. This new microplate-based method represents a fast, inexpensive and non-radioactive alternative to existing D-loop assays. It provides accurate kinetic analysis of strand assimilation in high-throughput and performs well with human RAD51 and Escherichia coli RecA protein. This advance will aid in both mechanistic studies of homologous recombination and drug screening programs.

Condensation of DNA—A putative obstruction for repair process in abasic clustered DNA damage

Clustered DNA damages are defined as two or more closely located DNA damage lesions that may be present within a few helical turns of the DNA double strand. These damages are potential signatures of ionizing radiation and are often found to be repair resistant. Types of damaged lesions frequently found inside clustered DNA damage sites include oxidized bases, abasic sites, nucleotide dimers, strand breaks or their complex combinations. In this study, we used a bistranded two-lesion abasic cluster DNA damage model to access the repair process of DNA in condensate form.Oligomer DNA duplexes (47bp) were designed to have two deoxyuridine in the middle of the sequences, three bases apart in opposite strands. The deoxyuridine residues were converted into abasic sites by treatment with UDG enzyme creating an abasic clustered damage site in a precise position in each of the single strand of the DNA duplex. This oligomer duplex having compatible cohesive ends was ligated to pUC19 plasmid, linearized with HindIII restriction endonuclease. The plasmid–oligomer conjugate was transformed into condensates by treating them with spermidine. The efficiency of strand cleavage action of ApeI enzyme on the abasic sites was determined by denaturing PAGE after timed incubation of the oligomer duplex and the oligomer–plasmid conjugate in presence and absence of spermidine. The efficiency of double strand breaks was determined similarly by native PAGE. Quantitative gel analysis revealed that rate of abasic site cleavage is reduced in the DNA condensates as compared to the oligomer DNA duplex or the linear ligated oligomer–plasmid conjugates. Generation of double strand break is significantly reduced also, suggesting that their creation is not proportionate to the number of abasic sites cleaved in the condensate model. All these suggest that the ApeI enzyme have difficulty to access the abasic sites located deep into the condensates leading to repair refractivity of the damages. In addition, we found that presence of a polyamine such as spermidine has no notable effect in the incision activity of ApeI enzyme in linear oligomer DNA duplexes in our experimental concentration.

Is duplex DNA a swollen random coil?

Direct observation of the Brownian motion of linear and circular duplex DNA in the chain-length range from about 5 to 300 kilo-basepairs (kbp), as reported by Smith, Perkins, and Chu, [Macromolecules, 1996, 29, 1372–1373] and by Robertson, Laib, and Smith, [Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 7310–7314] has indicated an effective mass-scaling exponent for diffusion, νH, of approximately 0.6, and this correlation was interpreted as implying that duplex DNA in this chain-length range can be described as a flexible polymer swollen by excluded volume interactions. However, we present computational evidence, based on our path-integration technique for computing transport properties, that indicates this inference needs to be reconsidered. Because of variable draining effects, the ratio Rh/Rg for random-coil polymers, including DNA, varies gradually throughout the chain-length range of interest, and does not stabilize to an asymptotic value until the chains are extremely long. This slow exponent crossover is particularly extended in stiff chains such as DNA. In particular, we find that if we take duplex DNA to be a worm-like chain comprised of a number of base pairs in the range between 5 and 300 kbp, then the effective exponent νH happens to be about 0.6, while the effective exponent, ν, for the radius of gyration, Rg, is about 0.52 and 0.51, with and without chain excluded volume, respectively. These results were obtained directly for a stiff lattice-chain model that agrees very well with the predictions of the continuum worm-like chain model. Based on these calculations and consistent experimental observations, we suggest that duplex DNA having a length below about 300 kbp is better described as a worm-like chain than a self-avoiding walk.