Enzyme-assisted high throughput sequencing of an expanded genetic alphabet at single base resolution.

With just four building blocks, low sequence information density, few functional groups, poor control over folding, and difficulties in forming compact folds, natural DNA and RNA have been disappointing platforms from which to evolve receptors, ligands, and catalysts. Accordingly, synthetic biology has created "artificially expanded genetic information systems" (AEGIS) to add nucleotides, functionality, and information density. With the expected improvements seen in AegisBodies and AegisZymes, the task for synthetic biologists shifts to developing for expanded DNA the same analytical tools available to natural DNA. Here we report one of these, an enzyme-assisted sequencing of expanded genetic alphabet (ESEGA) method to sequence six-letter AEGIS DNA. We show how ESEGA analyses this DNA at single base resolution, and applies it to optimized conditions for six-nucleotide PCR, assessing the fidelity of various DNA polymerases, and extending this to AEGIS components with functional groups. This supports the renewed exploitation of expanded DNA alphabets in biotechnology.

Establishing a biochemical understanding of the initiation of chromosome replication in bacteria.

In the mid-1950s, Arthur Kornberg elucidated the enzymatic synthesis of DNA by DNA polymerase, for which he was recognized with the 1959 Nobel Prize in Physiology or Medicine. He then identified many of the proteins that cooperate with DNA polymerase to replicate duplex DNA of small bacteriophages. However, one major unanswered problem was understanding the mechanism and control of the initiation of chromosome replication in bacteria. In a seminal paper in 1981, Fuller, Kaguni, and Kornberg reported the development of a cell-free enzyme system that could replicate DNA that was dependent on the bacterial origin of DNA replication, oriC. This advance opened the door to a flurry of discoveries and important papers that elucidated the process and control of initiation of chromosome replication in bacteria.

Synthetic molecular switches driven by DNA-modifying enzymes.

Taking inspiration from natural systems, in which molecular switches are ubiquitous in the biochemistry regulatory network, we aim to design and construct synthetic molecular switches driven by DNA-modifying enzymes, such as DNA polymerase and nicking endonuclease. The enzymatic treatments on our synthetic DNA constructs controllably switch ON or OFF the sticky end cohesion and in turn cascade to the structural association or disassociation. Here we showcase the concept in multiple DNA nanostructure systems with robust assembly/disassembly performance. The switch mechanisms are first illustrated in minimalist systems with a few DNA strands. Then the ON/OFF switches are realized in complex DNA lattice and origami systems with designated morphological changes responsive to the specific enzymatic treatments.

RAD18 O-GlcNAcylation promotes translesion DNA synthesis and homologous recombination repair.

RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the regulatory mechanism of O-linked N-acetylglucosamine (O-GlcNAc) modification governing RAD18 and its function during these processes remains unknown. Here, we report that human RAD18, can undergo O-GlcNAcylation at Ser130/Ser164/Thr468, which is important for optimal RAD18 accumulation at DNA damage sites. Mechanistically, abrogation of RAD18 O-GlcNAcylation limits CDC7-dependent RAD18 Ser434 phosphorylation, which in turn significantly reduces damage-induced PCNA monoubiquitination, impairs Polη focus formation and enhances UV sensitivity. Moreover, the ubiquitin and RAD51C binding ability of RAD18 at DNA double-strand breaks (DSBs) is O-GlcNAcylation-dependent. O-GlcNAcylated RAD18 promotes the binding of RAD51 to damaged DNA during HR and decreases CPT hypersensitivity. Our findings demonstrate a novel role of RAD18 O-GlcNAcylation in TLS and HR regulation, establishing a new rationale to improve chemotherapeutic treatment.

Primordial germ cell DNA demethylation and development require DNA translesion synthesis.

Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7 -/-) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.

Lead compound profiling for small molecule inhibitors of the REV1-CT/RIR Translesion synthesis Protein-Protein interaction.

Translesion synthesis (TLS) is a cellular mechanism through which actively replicating cells recruit specialized, low-fidelity DNA polymerases to damaged DNA to allow for replication past these lesions. REV1 is one of these TLS DNA polymerases that functions primarily as a scaffolding protein to organize the TLS heteroprotein complex and ensure replication occurs in the presence of DNA lesions. The C-Terminal domain of REV1 (REV1-CT) forms many protein-protein interactions (PPIs) with other TLS polymerases, making it essential for TLS function and a promising drug target for anti-cancer drug development. We utilized several lead identification strategies to identify various small molecules capable of disrupting the PPI between REV1-CT and the REV1 Interacting Regions (RIR) present in several other TLS polymerases. These lead compounds were profiled in several in vitro potency and PK assays to identify two scaffolds (1 and 6) as the most promising for further development. Both 1 and 6 synergized with cisplatin in a REV1-dependent fashion and demonstrated promising in vivo PK and toxicity profiles.

The Catalytic Activity of Human REV1 on Undamaged and Damaged DNA.

Eukaryotic REV1 serves as a scaffold protein for the coordination of DNA polymerases during DNA translesion synthesis. Besides this structural role, REV1 is a Y-family DNA polymerase with its own distributive deoxycytidyl transferase activity. However, data about the accuracy and efficiency of DNA synthesis by REV1 in the literature are contrasting. Here, we expressed and purified the full-length human REV1 from Saccharomyces cerevisiae and characterized its activity on undamaged DNA and a wide range of damaged DNA templates. We demonstrated that REV1 carried out accurate synthesis opposite 8-oxoG and O6-meG with moderate efficiency. It also replicated thymine glycol surprisingly well in an error-prone manner, but was blocked by the intrastrand 1,2-GG cisplatin crosslink. By using the 1,N6-ethenoadenine and 7-deaza-adenine lesions, we have provided biochemical evidence of the importance for REV1 functioning of the Hoogsteen face of template A, the second preferable template after G.

Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR.

Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10-5 bp-1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity - an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.

Sequential requirements for distinct Polθ domains during theta-mediated end joining.

DNA polymerase θ (Polθ) plays a central role in a DNA double-strand break repair pathway termed theta-mediated end joining (TMEJ). TMEJ functions by pairing short-sequence "microhomologies" (MHs) in single-stranded DNA at each end of a break and subsequently initiating DNA synthesis. It is not known how the Polθ helicase domain (HD) and polymerase domain (PD) operate to bring together MHs and facilitate repair. To resolve these transient processes in real time, we utilized in vitro single-molecule FRET approaches and biochemical analyses. We find that the Polθ-HD mediates the initial capture of two ssDNA strands, bringing them in close proximity. The Polθ-PD binds and stabilizes pre-annealed MHs to form a synaptic complex (SC) and initiate repair synthesis. Individual synthesis reactions show that Polθ is inherently non-processive, accounting for complex mutational patterns during TMEJ. Binding of Polθ-PD to stem-loop-forming sequences can substantially limit synapsis, depending on the available dNTPs and sequence context.

Recent advances in the synthesis of single-stranded DNA in vitro.

Single-stranded DNA (ssDNA) is the foundation of modern biology, with wide applications in gene editing, sequencing, DNA information storage, and materials science. However, synthesizing ssDNA with high efficiency, high throughput, and low error rate in vitro remains a major challenge. Various methods have been developed for ssDNA synthesis, and some significant results have been achieved. In this review, six main methods were introduced, including solid-phase oligonucleotide synthesis, terminal deoxynucleotidyl transferase-based ssDNA synthesis, reverse transcription, primer exchange reaction, asymmetric polymerase chain reaction, and rolling circle amplification. The advantages and limitations of each method were compared, as well as illustrate their representative achievements and applications. Especially, rolling circle amplification has received significant attention, including ssDNA synthesis, assembly, and application based on recent work. Finally, the future challenges and opportunities of ssDNA synthesis were summarized and discussed. Envisioning the development of new methods and significant progress will be made in the near future with the efforts of scientists around the world.

Melanoma-Derived DNA Polymerase Theta Variants Exhibit Altered DNA Polymerase Activity.

DNA polymerase θ (Pol θ or POLQ) is primarily involved in repairing double-stranded breaks in DNA through an alternative pathway known as microhomology-mediated end joining (MMEJ) or theta-mediated end joining (TMEJ). Unlike other DNA repair polymerases, Pol θ is thought to be highly error-prone yet critical for cell survival. We have identified several POLQ gene variants from human melanoma tumors that experience altered DNA polymerase activity, including a propensity for incorrect nucleotide selection and reduced polymerization rates compared to WT Pol θ. Variants are 30-fold less efficient at incorporating a nucleotide during repair and up to 70-fold less accurate at selecting the correct nucleotide opposite a templating base. This suggests that aberrant Pol θ has reduced DNA repair capabilities and may also contribute to increased mutagenesis. Moreover, the variants were identified in established tumors, suggesting that cancer cells may use mutated polymerases to promote metastasis and drug resistance.

Discovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors.

The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4-6 nM IC50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi.

Combination of ganciclovir and trifluridine prevents drug-resistance emergence in HSV-1.

Ocular herpes simplex virus 1 (HSV-1) infections can lead to visual impairment. Long-term acyclovir (ACV) prophylaxis reduces the frequency of recurrences but is associated with drug resistance. Novel therapies are needed to treat drug-resistant HSV-1 infections. Here, we describe the effects of trifluridine (TFT) in combination with ACV or ganciclovir (GCV) on HSV-1 replication and drug-resistance emergence. Wild-type HSV-1 was grown under increasing doses of one antiviral (ACV, GCV, or TFT) or combinations thereof (ACV + TFT or GCV + TFT). Virus cultures were analyzed by Sanger sequencing and deep sequencing of the UL23 [thymidine kinase (TK)] and UL30 [DNA polymerase (DP)] genes. The phenotypes of novel mutations were determined by cytopathic effect reduction assays. TFT showed overall additive anti-HSV-1 activity with ACV and GCV. Five passages under ACV, GCV, or TFT drug pressure gave rise to resistance mutations, primarily in the TK. ACV + TFT and GCV + TFT combinatory pressure induced mutations in the TK and DP. The DP mutations were mainly located in terminal regions, outside segments that typically carry resistance mutations. TK mutations (R163H, A167T, and M231I) conferring resistance to all three nucleoside analogs (ACV, TFT, and GCV) emerged under ACV, TFT, ACV + TFT pressure and under GCV + TFT pressure initiated from suboptimal drug concentrations. However, higher doses of GCV and TFT prevented drug resistance in the resistance selection experiments. In summary, we identified novel mutations conferring resistance to nucleoside analogs, including TFT, and proposed that GCV + TFT combination therapy may be an effective strategy to prevent the development of drug resistance.

Zebrafish polg2 knock-out recapitulates human POLG-disorders; implications for drug treatment.

The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.

Expedient production of site specifically nucleobase-labelled or hypermodified RNA with engineered thermophilic DNA polymerases.

Innovative approaches to controlled nucleobase-modified RNA synthesis are urgently needed to support RNA biology exploration and to synthesize potential RNA therapeutics. Here we present a strategy for enzymatic construction of nucleobase-modified RNA based on primer-dependent engineered thermophilic DNA polymerases - SFM4-3 and TGK. We demonstrate introduction of one or several different base-modified nucleotides in one strand including hypermodified RNA containing all four modified nucleotides bearing four different substituents, as well as strategy for primer segment removal. We also show facile site-specific or segmented introduction of fluorophores or other functional groups at defined positions in variety of RNA molecules, including structured or long mRNA. Intriguing translation efficacy of single-site modified mRNAs underscores the necessity to study isolated modifications placed at designer positions to disentangle their biological effects and enable development of improved mRNA therapeutics. Our toolbox paves the way for more precise dissecting RNA structures and functions, as well as for construction of diverse types of base-functionalized RNA for therapeutic applications and diagnostics.

An acid-responsive DNA hydrogel-mediated cascaded enzymatic nucleic acid amplification system for the sensitive imaging of alkaline phosphatase in living cells.

Alkaline phosphatase (ALP) is a class of hydrolase that catalyzes the dephosphorylation of phosphorylated species in biological tissues, playing an important role in many physiological and pathological processes. Sensitive imaging of ALP activity in living cells is contributory to the research on these processes. Herein, we propose an acid-responsive DNA hydrogel to deliver a cascaded enzymatic nucleic acid amplification system into cells for the sensitive imaging of intracellular ALP activity. The DNA hydrogel is formed by two kinds of Y-shaped DNA monomers and acid-responsive cytosine-rich linkers. The amplification system contained Bst DNA polymerase (Bst DP), Nt.BbvCI endonuclease, a Recognition Probe (RP, containing a DNAzyme sequence, a Nt.BbvCI recognition sequence, and a phosphate group at the 3'-end), and a Signal Probe (SP, containing a cleavage site for DNAzyme, Cy3 and BHQ2 at the two ends). The amplification system was trapped into the DNA hydrogel and taken up by cells, and the cytosine-rich linkers folded into a quadruplex i-motif in the acidic lysosomes, leading to the collapse of the hydrogel and releasing the amplification system. The phosphate groups on RPs were recognized and removed by the target ALP, triggering a polymerization-nicking cycle to produce large numbers of DNAzyme sequences, which then cleaved multiple SPs, restoring Cy3 fluorescence to indicate the ALP activity. This strategy achieved sensitive imaging of ALP in living HeLa, MCF-7, and NCM460 cells, and realized the sensitive detection of ALP in vitro with a detection limit of 2.0 × 10-5 U mL-1, providing a potential tool for the research of ALP-related physiological and pathological processes.

Xanthomonas Phage PBR31: Classifying the Unclassifiable.

The ability of bacteriophages to destroy bacteria has made them the subject of extensive research. Interest in bacteriophages has recently increased due to the spread of drug-resistant bacteria, although genomic research has not kept pace with the growth of genomic data. Genomic analysis and, especially, the taxonomic description of bacteriophages are often difficult due to the peculiarities of the evolution of bacteriophages, which often includes the horizontal transfer of genes and genomic modules. The latter is particularly pronounced for temperate bacteriophages, which are capable of integration into the bacterial chromosome. Xanthomonas phage PBR31 is a temperate bacteriophage, which has been neither described nor classified previously, that infects the plant pathogen Xanthomonas campestris pv. campestris. Genomic analysis, including phylogenetic studies, indicated the separation of phage PBR31 from known classified bacteriophages, as well as its distant relationship with other temperate bacteriophages, including the Lederbervirus group. Bioinformatic analysis of proteins revealed distinctive features of PBR31, including the presence of a protein similar to the small subunit of D-family DNA polymerase and advanced lysis machinery. Taxonomic analysis showed the possibility of assigning phage PBR31 to a new taxon, although the complete taxonomic description of Xanthomonas phage PBR31 and other related bacteriophages is complicated by the complex evolutionary history of the formation of its genome. The general biological features of the PBR31 phage were analysed for the first time. Due to its presumably temperate lifestyle, there is doubt as to whether the PBR31 phage is appropriate for phage control purposes. Bioinformatics analysis, however, revealed the presence of cell wall-degrading enzymes that can be utilised for the treatment of bacterial infections.

Discovery of a potent inhibitor, D-132, targeting AsfvPolX, via protein-DNA complex-guided pharmacophore screening and in vitro molecular characterizations.

The heightened transmissibility and capacity of African swine fever virus (ASFV) induce fatal diseases in domestic pigs and wild boars, posing significant economic repercussions and global threats. Despite extensive research efforts, the development of potent vaccines or treatments for ASFV remains a persistent challenge. Recently, inhibiting the AsfvPolX, a key DNA repair enzyme, emerges as a feasible strategy to disrupt viral replication and control ASFV infections. In this study, a comprehensive approach involving pharmacophore-based inhibitor screening, coupled with biochemical and biophysical analyses, were implemented to identify, characterize, and validate potential inhibitors targeting AsfvPolX. The constructed pharmacophore model, Phar-PolX-S, demonstrated efficacy in identifying a potent inhibitor, D-132 (IC50 = 2.8 ± 0.2 µM), disrupting the formation of the AsfvPolX-DNA complex. Notably, D-132 exhibited strong binding to AsfvPolX (KD = 6.9 ± 2.2 µM) through a slow-on-fast-off binding mechanism. Employing molecular modeling, it was elucidated that D-132 predominantly binds in-between the palm and finger domains of AsfvPolX, with crucial residues (R42, N48, Q98, E100, F102, and F116) identified as hotspots for structure-based inhibitor optimization. Distinctively characterized by a 1,2,5,6-tetrathiocane with modifications at the 3 and 8 positions involving ethanesulfonates, D-132 holds considerable promise as a lead compound for the development of innovative agents to combat ASFV infections.