DNA Termini Research Articles

Human 8-Oxoguanine Glycosylase OGG1 Cleaves Abasic Sites and Covalently Conjugates to 3'-DNA Termini via Cysteine and Histidine Addition.

8-Oxoguanine glycosylase 1 (OGG1) repairs the major oxidative DNA damage, 8-oxo-2'-deoxyguanosine. It has been reported that OGG1 incises the most frequently formed DNA lesion, apurinic/apyrimidinic (AP) site, and in the process a stable DNA-OGG1 cross-link is formed. However, the chemical structure of the adduct is not characterized. Here, we report that DNA-OGG1 cross-links result from cysteine and histidine addition to incised AP sites at 3'-DNA termini.

Fluorocarbon-DNA Conjugates for Enhanced Cellular Delivery: Formation of a Densely Packed DNA Nano-Assembly.

Forming nano-assemblies is essential for delivering DNA conjugates into cells, with the DNA density in the nano-assembly playing an important role in determining the uptake efficiency. In this study, we developed a strategy for the facile synthesis of DNA strands bearing perfluoroalkyl (RF) groups (RF-DNA conjugates) and investigated how they affect cellular uptake. An RF-DNA conjugate bearing a long RF group at the DNA terminus forms a nano-assembly with a high DNA density, which results in greatly enhanced cellular uptake. The uptake mechanism is mediated by clathrin-dependent endocytosis. The use of RF groups to densely assemble negatively charged DNA is a useful strategy for designing drug delivery carriers.

Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells.

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-stranded breaks (DSBs) in vertebrates. However, due to challenges in detecting DSBs in living cells, the repair capacity of the NHEJ pathway is unknown. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed ~115Å apart in one of several long-range synaptic complexes before transitioning into a short-range synaptic complex that juxtaposes DNA ends to facilitate ligation. The transition from long-range to short-range synaptic complexes involves both conformational and compositional changes of the NHEJ factors bound to the DNA break. Importantly, it is unclear how NHEJ proceeds in vivo because of the challenges involved in analyzing recruitment of NHEJ factors to DSBs over time in living cells. Here, we develop a new approach to study the temporal and compositional dynamics of NHEJ complexes using live cell single-molecule imaging. Our results provide direct evidence for stepwise maturation of the NHEJ complex, pinpoint key regulatory steps in NHEJ progression, and define the overall repair capacity NHEJ in living cells.

Kinetic analysis of T4 polynucleotide kinase via isothermal titration calorimetry

T4 polynucleotide kinase (T4 PNK) phosphorylates the 5′-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3′-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3′-monophosphate (3′-AMP) to adenosine-3′,5′-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3′-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, KM, was 13 μM, and the turnover number, kcat, was 8 s−1. The effect of inhibitors was investigated using pyrophosphate (PPi). PPi caused a dose-dependent decrease in the apparent kcat and increase in the apparent KM under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3′-AMP were determined to be −25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach.

Changing structures, changing paradigms: NELF helps regulate paused or elongating RNA polymerase II

Using cryo-EM and biochemical methods, Su and Vos1 discover an alternative NELF structural state that enables transcription and switches NELF-RNA polymerase II (RNAPII) compatibility with other RNAPII-associated factors that regulate pausing, elongation, termination, and transcription-coupled DNA repair.

Structures of LIG1 provide a mechanistic basis for understanding a lack of sugar discrimination against a ribonucleotide at the 3'-end of nick DNA

Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of the broken strand breaks in the three-steps of ligation reaction. LIG1 can tolerate the RNA strand at the upstream of the nick, yet an atomic insight into the sugar discrimination mechanism by LIG1 against a ribonucleotide at the 3'-terminus of nick DNA is unknown. Here, we determined X-ray structures of LIG1/3'-RNA-DNA hybrids and captured the ligase during pre- and post-step 3 the ligation reaction. Furthermore, the overlays of 3'-rA:T and 3'-rG:C step 3 structures with step 2 structures of canonical 3'-dA:T and 3'-dG:C uncover a network of LIG1/DNA interactions through Asp570 and Arg871 side chains with 2'-OH of the ribose at nick showing a final phosphodiester bond formation and the other ligase active site residues surrounding the AMP site. Finally, we demonstrated that LIG1 can ligate the nick DNA substrates with preinserted 3'-ribonucleotides as efficient as Watson-Crick base-paired ends in vitro. Together, our findings uncover a novel atomic insight into a lack of sugar discrimination by LIG1 and the impact of improper sugar on the nick sealing of ribonucleotides at the last step of DNA replication and repair.

Binding to nucleosome poises human SIRT6 for histone H3 deacetylation.

Sirtuin 6 (SIRT6) is an NAD+-dependent histone H3 deacetylase that is prominently found associated with chromatin, attenuates transcriptionally active promoters and regulates DNA repair, metabolic homeostasis and lifespan. Unlike other sirtuins, it has low affinity to free histone tails but demonstrates strong binding to nucleosomes. It is poorly understood how SIRT6 docking on nucleosomes stimulates its histone deacetylation activity. Here, we present the structure of human SIRT6 bound to a nucleosome determined by cryogenic electron microscopy. The zinc finger domain of SIRT6 associates tightly with the acidic patch of the nucleosome through multiple arginine anchors. The Rossmann fold domain binds to the terminus of the looser DNA half of the nucleosome, detaching two turns of the DNA from the histone octamer and placing the NAD+ binding pocket close to the DNA exit site. This domain shows flexibility with respect to the fixed zinc finger and moves with, but also relative to, the unwrapped DNA terminus. We apply molecular dynamics simulations of the histone tails in the nucleosome to show that in this mode of interaction, the active site of SIRT6 is perfectly poised to catalyze deacetylation of the H3 histone tail and that the partial unwrapping of the DNA allows even lysines close to the H3 core to reach the enzyme.

Binding to nucleosome poises human SIRT6 for histone H3 deacetylation

Sirtuin 6 (SIRT6) is an NAD+-dependent histone H3 deacetylase that is prominently found associated with chromatin, attenuates transcriptionally active promoters and regulates DNA repair, metabolic homeostasis and lifespan. Unlike other sirtuins, it has low affinity to free histone tails but demonstrates strong binding to nucleosomes. It is poorly understood how SIRT6 docking on nucleosomes stimulates its histone deacetylation activity. Here, we present the structure of human SIRT6 bound to a nucleosome determined by cryogenic electron microscopy. The zinc finger domain of SIRT6 associates tightly with the acidic patch of the nucleosome through multiple arginine anchors. The Rossmann fold domain binds to the terminus of the looser DNA half of the nucleosome, detaching two turns of the DNA from the histone octamer and placing the NAD+ binding pocket close to the DNA exit site. This domain shows flexibility with respect to the fixed zinc finger and moves with, but also relative to, the unwrapped DNA terminus. We apply molecular dynamics simulations of the histone tails in the nucleosome to show that in this mode of interaction, the active site of SIRT6 is perfectly poised to catalyze deacetylation of the H3 histone tail and that the partial unwrapping of the DNA allows even lysines close to the H3 core to reach the enzyme.

Mechanism of Viral DNA Packaging in Phage T4 Using Single-Molecule Fluorescence Approaches.

In all tailed phages, the packaging of the double-stranded genome into the head by a terminase motor complex is an essential step in virion formation. Despite extensive research, there are still major gaps in the understanding of this highly dynamic process and the mechanisms responsible for DNA translocation. Over the last fifteen years, single-molecule fluorescence technologies have been applied to study viral nucleic acid packaging using the robust and flexible T4 in vitro packaging system in conjunction with genetic, biochemical, and structural analyses. In this review, we discuss the novel findings from these studies, including that the T4 genome was determined to be packaged as an elongated loop via the colocalization of dye-labeled DNA termini above the portal structure. Packaging efficiency of the TerL motor was shown to be inherently linked to substrate structure, with packaging stalling at DNA branches. The latter led to the design of multiple experiments whose results all support a proposed torsional compression translocation model to explain substrate packaging. Evidence of substrate compression was derived from FRET and/or smFRET measurements of stalled versus resolvase released dye-labeled Y-DNAs and other dye-labeled substrates relative to motor components. Additionally, active in vivo T4 TerS fluorescent fusion proteins facilitated the application of advanced super-resolution optical microscopy toward the visualization of the initiation of packaging. The formation of twin TerS ring complexes, each expected to be ~15 nm in diameter, supports a double protein ring-DNA synapsis model for the control of packaging initiation, a model that may help explain the variety of ring structures reported among pac site phages. The examination of the dynamics of the T4 packaging motor at the single-molecule level in these studies demonstrates the value of state-of-the-art fluorescent tools for future studies of complex viral replication mechanisms.

Turn-on fluorescent nanoprobe for ATP detection based on DNA-templated silver nanoclusters.

A turn-on fluorescence nanoprobe was constructed for the determination of adenosine 5'-triphosphate (ATP) based on DNA-templated silver nanoclusters (DNA-AgNCs). The significant enhancement fluorescence intensity of DNA-AgNCs in the presence of ATP is due to the high special binding affinity between ATP and the aptamer, resulting in the environment of DNA-AgNCs with darkish fluorescence lying at one terminus of DNA slightly altering owing to the change of ATP aptamer conformation. A good linear range runs from 9 to 24 mM with a satisfactory detection limit of 3 μM. Furthermore, the proposed nanoprobe exhibited good performance for ATP detection in diluted fetal bovine serum.

Mechanisms of Cancer-Resistant Mammals

Cancer is a medical disorder when a few of the body’s cells multiply uncontrollably and extend in a considerable amount of time. Researchers are trying everything to find a solution for cancer. This review is mainly focused on elephants, bowhead whales, naked mole rats and their inner mechanisms, which make them resistant to cancer. Statistically, larger animals such as elephants have much more cells then human, which means that they ought to have more cases of cancer, but there is an inner mechanism, which works in these animals and makes them cancer resistant. Elephants possess unique p53 genes with 20 copies, while people only have one. If cells are severely damaged, the p53 genes instructs the cells to be destroyed due to potential cancer risk. This process appeared to cause them to activate the LIF6 gene, and that leads to apoptosis of shattered cells. Bowhead whales are able to repair double-strand breaks more accurately and more efficiently. Bowhead whales are less likely than humans to create removals when repairing mismatched DNA termini. Naked mole rat cells are immune to cancer as they are more susceptible to early and firsthand suppression, which is a mechanism that prevents tumors from forming. Unfortunately, humans are still clueless about how to transfer the cancer preventing methods which exist in these three magical organisms into human. Hopefully, with the rapid development of technology and advances in the biomedical field, one day scientists will be able to put these theories into practice.

Structural basis for DNA proofreading

DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base translocates from the polymerase to the exonuclease site and the corrected DNA terminus returns has remained elusive. Here, we present an ensemble of nine high-resolution structures representing human mitochondrial DNA polymerase Gamma, Polγ, captured during consecutive proofreading steps. The structures reveal key events, including mismatched base recognition, its dissociation from the polymerase site, forward translocation of DNAP, alterations in DNA trajectory, repositioning and refolding of elements for primer separation, DNAP backtracking, and displacement of the mismatched base into the exonuclease site. Altogether, our findings suggest a conserved ‘bolt-action’ mechanism of proofreading based on iterative cycles of DNAP translocation without dissociation from the DNA, facilitating primer transfer between catalytic sites. Functional assays and mutagenesis corroborate this mechanism, connecting pathogenic mutations to crucial structural elements in proofreading steps.

Quantitative analysis of preferential utilization of AAV ITR as the packaging terminal signal.

Genetic engineering advances have led to recombinant adeno-associated virus (rAAV) becoming an invaluable tool for the development of effective gene therapies. The production of rAAV is susceptible to off-target heterogeneous packaging, the effects of which are still being understood. Here, rAAV vectors with four-genome lengths were produced using both adherent and suspension HEK293 cells to understand the 5'ITR termination. AAV8 vectors were produced from the human FVIII plasmid for a full-length cargo of 4,707 nucleotides with specific truncations, creating smaller genomes. Conventionally, rAAV is characterized by differentiating empty capsids from full capsids, but for this work, that description is incomplete. The small genomes in this study were characterized by charge detection-mass spectrometry (CD-MS). Using CD-MS, packaged genomes in the range conventionally attributed to partials were resolved and quantified. In addition, alkaline gels and qPCR were used to assess the identity of the packaged genomes. Together, these results showed a propensity for unit-length genomes to be encapsidated. Packaged genomes occurred as replication intermediates emanating from the 5'ITR, indicating that HEK293 cells prefer unit-length genomes as opposed to the 5'ITR termination and heterogeneous DNA packaging observed previously from Sf9 cell systems. As both manufacturing processes are used and continually assessed to produce clinical material, such an understanding will benefit rAAV design for basic research and gene therapy.

Extraordinarily Stable Hairpin-Based Biosensors for Rapid Detection of DNA Ligases.

DNA ligases are essential enzymes involved in DNA replication and repair processes in all organisms. These enzymes seal DNA breaks by catalyzing the formation of phosphodiester bonds between juxtaposed 5' phosphate and 3' hydroxyl termini in double-stranded DNA. In addition to their critical roles in maintaining genomic integrity, DNA ligases have been recently identified as diagnostic biomarkers for several types of cancers and recognized as potential drug targets for the treatment of various diseases. Although DNA ligases are significant in basic research and medical applications, developing strategies for efficiently detecting and precisely quantifying these crucial enzymes is still challenging. Here, we report our design and fabrication of a highly sensitive and specific biosensor in which a stable DNA hairpin is utilized to stimulate the generation of fluorescence signals. This probe is verified to be stable under a wide range of experimental conditions and exhibits promising performance in detecting DNA ligases. We anticipate that this hairpin-based biosensor will significantly benefit the development of new targeting strategies and diagnostic tools for certain diseases.

Centromere plasticity with evolutionary conservation and divergence uncovered by wheat 10+ genomes.

Centromeres are the chromosomal regions that play a crucial role in maintaining genomic stability. The underling highly repetitive DNA sequences can evolve quickly in most eukaryotes, and promote karyotype evolution. Despite their variability, it is not fully understood how these widely variable sequences ensure the homeostasis of centromere function. In this study, we investigated the genetics and epigenetics of centromeres in a population of wheat lines from global breeding programs. We captured a high degree of sequences, positioning, and epigenetic variations in the large and complex wheat centromeres. We found that the most CENH3-associated repeats are Cereba element of retrotransposons and exhibit phylogenetic homogenization across different wheat lines, but the less-associated repeat sequences diverge on their own way in each wheat line, implying specific mechanisms for selecting certain repeat types as functional core centromeres. Furthermore, we observed that CENH3 nucleosome structures display looser wrapping of DNA termini on complex centromeric repeats, including the repositioned centromeres. We also found that strict CENH3 nucleosome positioning and intrinsic DNA features play a role in determining centromere identity among different lines. Specific non-B form DNAs were substantially associated with CENH3 nucleosomes for the repositioned centromeres. These findings suggest that multiple mechanisms were involved in the adaptation of CENH3 nucleosomes that can stabilize centromeres. Ultimately, we proposed a remarkable epigenetic plasticity of centromere chromatin within the diverse genomic context, and the high robustness is crucial for maintaining centromere function and genome stability in wheat 10+ lines as a result of past breeding selections.

Peroxidase-Mimicking Activity of Nanoceria for Label-Free Colorimetric Assay for Exonuclease III Activity.

We present a novel label-free colorimetric method for detecting exonuclease III (Exo III) activity using the peroxidase-mimicking activity of cerium oxide nanoparticles (nanoceria). Exo III, an enzyme that specifically catalyzes the stepwise removal of mononucleotides from the 3'-OH termini of double-stranded DNA, plays a significant role in various cellular and physiological processes, including DNA proofreading and repair. Malfunctions of Exo III have been associated with increased cancer risks. To assay the activity of Exo III, we applied the previous reports in that the peroxidase-mimicking activity of nanoceria is inhibited due to the aggregation induced by the electrostatic attraction between DNA and nanoceria. In the presence of Exo III, the substrate DNA (subDNA), which inhibits nanoceria's activity, is degraded, thereby restoring the peroxidase-mimicking activity of nanoceria. Consequently, the 3,3',5,5'-tetramethylbenzidine (TMB) substrate is oxidized, leading to a color change from colorless to blue, along with an increase in the absorbance intensity. This approach enabled us to reliably detect Exo III at a limit of detection (LOD) of 0.263 units/mL across a broad dynamic range from 3.1 to 400 units/mL, respectively, with an outstanding specificity. Since this approach does not require radiolabels, complex DNA design, or sophisticated experimental techniques, it provides a simpler and more feasible alternative to standard methods.

Prokaryotic Gabija complex senses and executes nucleotide depletion and DNA cleavage for antiviral defense

The Gabija complex is a prokaryotic antiviral system consisting of the GajA and GajB proteins. GajA was identified as a DNA nicking endonuclease but the functions of GajB and the complex remain unknown. Here, we show that synergy between GajA-mediated DNA cleavage and nucleotide hydrolysis by GajB initiates efficient abortive infection defense against virulent bacteriophages. The antiviral activity of GajA requires GajB, which senses DNA termini produced by GajA to hydrolyze (d)A/(d)GTP, depleting essential nucleotides. This ATPase activity of Gabija complex is only activated upon DNA binding. GajA binds to GajB to form stable complexes invivo and invitro. However, a functional Gabija complex requires a molecular ratio between GajB and GajA below 1:1, indicating stoichiometric regulation of the DNA/nucleotide processing complex. Thus, the Gabija system exhibits distinct and efficient antiviral defense through sequential sensing and activation of nucleotide depletion and DNA cleavage, causing a cascade suicide effect.

Synthesis and Excision Repair of Site-Specific 3'-End DNA-Histone Cross-Links Derived from Abasic Sites.

Histones catalyze the DNA strand incision at apurinic/apyrimidinic (AP) sites accompanied by formation of reversible but long-lived DNA-protein cross-links (DPCs) at 3'-DNA termini within single-strand breaks. These DPCs need to be removed because 3'-hydroxyl is required for gap-filling DNA repair synthesis but are challenging to study because of their reversible nature. Here we report a chemical approach to synthesize stable and site-specific 3'-histone-DPCs and their repair by three nucleases, human AP endonuclease 1, tyrosyl-DNA phosphodiesterase 1, and three-prime repair exonuclease 1. Our method employs oxime ligation to install an alkyne to 3'-DNA terminus, genetic incorporation of an azidohomoalanine to histone H4 at a defined position, and click reaction to conjugate DNA to H4 site-specifically. Using these model DPC substrates, we demonstrated that the DPC repair efficiency is highly affected by the local protein environment, and prior DPC proteolysis facilitates the repair.

Corruption of DNA end-joining in mammalian chromosomes by progerin expression

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging and a life expectancy of about 14 years. HGPS is commonly caused by a point mutation in the LMNA gene which codes for lamin A, an essential component of the nuclear lamina. The HGPS mutation alters splicing of the LMNA transcript, leading to a truncated, farnesylated form of lamin A termed "progerin." Progerin is also produced in small amounts in healthy individuals by alternative splicing of RNA and has been implicated in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs), suggesting alteration of DNA repair. DSB repair normally occurs by either homologous recombination (HR), an accurate, templated form of repair, or by nonhomologous end-joining (NHEJ), a non-templated rejoining of DNA ends that can be error-prone; however a good portion of NHEJ events occurs precisely with no alteration to joined sequences. Previously, we reported that over-expression of progerin correlated with increased NHEJ relative to HR. We now report on progerin’s impact on the nature of DNA end-joining. We used a model system involving a DNA end-joining reporter substrate integrated into the genome of cultured thymidine kinase-deficient mouse fibroblasts. Some cells were engineered to express progerin. Two closely spaced DSBs were induced in the integrated substrate through expression of endonuclease I-SceI, and DSB repair events were recovered through selection for thymidine kinase function. DNA sequencing revealed that progerin expression correlated with a significant shift away from precise end-joining between the two I-SceI sites and toward imprecise end-joining. Additional experiments revealed that progerin did not reduce HR fidelity. Our work suggests that progerin suppresses interactions between complementary sequences at DNA termini, thereby shifting DSB repair toward low-fidelity DNA end-joining and perhaps contributing to accelerated and normal aging through compromised genome stability.

TtAgo sensor for the sensitive and rapid detection of T4 polynucleotide kinase activity

As a next-generation biosensing technology, the application of Argonaute for analytical science research has attracted considerable attention. A Thermus thermophilus Argonaute (TtAgo)-based sensor is presented for detecting T4 polynucleotide kinase (PNK) activity considering that TtAgo’s multiple-turnover catalytic activity can be activated by binding to a 5′-phosphate termini of single-stranded DNA (ssDNA) for the cleavage of complementary strand. PNK catalyzes the phosphorylation of 5′-hydroxyl termini of ssDNA, and the phosphorylation product termed guide can activate the TtAgo-mediated complementary ssDNA reporter cleavage to release fluorescence signal. For the PNK activity monitoring, the developed method shows good analytic performance with a linear range of 0.0010–2 U mL−1 and a detection limit of 0.0010 U mL−1 without the use of a preamplification reaction. This method is successfully applied for the analysis of HeLa cells at a single-cell level. The developed method shows several advantages, including high sensitivity, low cost, facile operation, and has remarkable potential application for early disease diagnosis, drug screening, and clinical treatment.