The molecular basis and disease relevance of non-homologous DNA end joining.

Non-homologous DNA end joining (NHEJ) is the predominant repair mechanism of any type of DNA double-strand break (DSB) during most of the cell cycle and is essential for the development of antigen receptors. Defects in NHEJ result in sensitivity to ionizing radiation and loss of lymphocytes. The most critical step of NHEJ is synapsis, or the juxtaposition of the two DNA ends of a DSB, because all subsequent steps rely on it. Recent findings show that, like the end processing step, synapsis can be achieved through several mechanisms. In this Review, we first discuss repair pathway choice between NHEJ and other DSB repair pathways. We then integrate recent insights into the mechanisms of NHEJ synapsis with updates on other steps of NHEJ, such as DNA end processing and ligation. Finally, we discuss NHEJ-related human diseases, including inherited disorders and neoplasia, which arise from rare failures at different NHEJ steps.

Novel Flavonoid Photoswitchable "Turn-On" Fluorescent Chemosensors: Synthesis of Bromo Flavonols for Nanomolar Aluminum Ion Detection and Cellular Imaging, among Other Applications.

Aluminum (Al), a non-essential element in living systems, can potentially cause chronic toxicity. Therefore, it is crucial to have a specific and sensitive method for detecting Al3+ in order to assess its risk to life. In this study, we designed and synthesized a novel fluorescent probe (IV) based on bromoflavonol. Upon binding to Al3+, probe IV exhibits a blue shift in emission and enhanced fluorescence, making it suitable for Al3+ detection. Our UV-Vis absorption and fluorescence emission spectra demonstrate that probe IV has high selectivity and sensitivity towards Al3+ while being immune to interference from other metal ions. Through fluorescence titration, we determined that the detection limit (LOD) of probe IV for Al3+ is 1.8 × 10-8 mol/L. Job's curve and 1 H NMR titration further confirmed a 1:1 binding stoichiometry between probe IV and Al3+. Additionally, using DFT (Density Functional Theory), we calculated the energy gap difference between IV and IV + Al3+ and found that the complex formed by probe IV and Al3+ is more stable than IV alone. We successfully detected Al3+ in tap water and river water from the middle regions of the Han River, achieving recoveries of over 96% using this probe. This demonstrates its potential for quantitative detection of Al3+ in environmental water samples. Moreover, we successfully used the probe for imaging Al3+ in MG63 cells, suggesting its potential application in biological imaging.

Polymerase µ in non-homologous DNA end joining: importance of the order of arrival at a double-strand break in a purified system.

During non-homologous DNA end joining (NHEJ), bringing two broken dsDNA ends into proximity is an essential prerequisite for ligation by XRCC4:Ligase IV (X4L4). This physical juxtaposition of DNA ends is called NHEJ synapsis. In addition to the key NHEJ synapsis proteins, Ku, X4L4, and XLF, it has been suggested that DNA polymerase mu (pol µ) may also align two dsDNA ends into close proximity for synthesis. Here, we directly observe the NHEJ synapsis by pol µ using a single molecule FRET (smFRET) assay where we can measure the duration of the synapsis. The results show that pol µ alone can mediate efficient NHEJ synapsis of 3' overhangs that have at least 1 nt microhomology. The abundant Ku protein in cells limits the accessibility of pol µ to DNA ends with overhangs. But X4L4 can largely reverse the Ku inhibition, perhaps by pushing the Ku inward to expose the overhang for NHEJ synapsis. Based on these studies, the mechanistic flexibility known to exist at other steps of NHEJ is now also apparent for the NHEJ synapsis step.

NAD+ is not utilized as a co-factor for DNA ligation by human DNA ligase IV.

As nucleotidyl transferases, formation of a covalent enzyme-adenylate intermediate is a common first step of all DNA ligases. While it has been shown that eukaryotic DNA ligases utilize ATP as the adenylation donor, it was recently reported that human DNA ligase IV can also utilize NAD+ and, to a lesser extent ADP-ribose, as the source of the adenylate group and that NAD+, unlike ATP, enhances ligation by supporting multiple catalytic cycles. Since this unexpected finding has significant implications for our understanding of the mechanisms and regulation of DNA double strand break repair, we attempted to confirm that NAD+ and ADP-ribose can be used as co-factors by human DNA ligase IV. Here, we provide evidence that NAD+ does not enhance ligation by pre-adenylated DNA ligase IV, indicating that this co-factor is not utilized for re-adenylation and subsequent cycles of ligation. Moreover, we find that ligation by de-adenylated DNA ligase IV is dependent upon ATP not NAD+ or ADP-ribose. Thus, we conclude that human DNA ligase IV cannot use either NAD+ or ADP-ribose as adenylation donor for ligation.

Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine.

The gene- or fragment-specific detection of newly recognized deoxyribonucleic acid (DNA) base 5-hydroxymethylcytosine (5hmC) will provide insights into its critical functions in development and diseases, and is also important for screening 5hmC-rich genes as an indicator of epigenetic states, pathogenic processes and pharmacological responses. Current analytical technologies for gene-specific detection of 5hmC are heavily dependent on glucosylated 5hmC-resistant restriction endonuclease cleavage. Here, we find that boronic acid (BA) can inhibit the amplification activity of Taq DNA polymerase for replicating glucosylated 5hmC bases in template DNA by interacting with their glucose moiety. On the basis of this finding, we propose for the first time a BA-mediated polymerase chain reaction (PCR) assay for rapid and sensitive detection of gene- or fragment-specific 5hmC without restriction-assay-like sequence limitations. To optimize the BA-mediated PCR assay, we further tested BA derivatives and show that one BA derivative, 2-(2'-chlorobenzyloxy) phenylboronic acid, displays the highest inhibitory efficiency. Using the optimized assay, we demonstrate the enrichment of 5hmC in an intron region of Pax5 gene (a member of the paired box family of transcription factors) in mouse embryonic stem cells. Our work potentially opens a new way for the screening and identification of 5hmC-rich genes and for high throughput analysis of 5hmC in mammalian cells.

Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism.

DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5 mC to 5 hmC in vivo, and increase 5 hmC in 5751 genes in cells. 5 hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5 mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5 hmC increase, and a delivery of labile iron increased the 5 hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5 hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules.

Electrophoretic behavior of DNA-methyl-CpG-binding domain protein complexes revealed by capillary electrophoreses laser-induced fluorescence.

The free solution electrophoretic behavior of DNA-protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA-protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA-methyl-CpG-binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA-protein complexes. This method is based on the effective separation of free DNA and DNA-protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide-coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA-MBD2b complexes, we found that the values of (µ0 /µ)-1 were directly proportional to the charge-to-mass ratios of formed complexes, where the µ0 and µ are the mobility of free DNA probe and DNA-protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge-to-mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA-binding properties of the purified MBD2b protein and help to analyze other DNA-protein complexes. Additionally, this study enhances the understanding of the influence of the charge-to-mass ratios of formed DNA-protein complexes on their separation and electrophoretic behaviors.

Interplay of binding stoichiometry and recognition specificity for the interaction of MBD2b protein and methylated DNA revealed by affinity capillary electrophoresis coupled with laser-induced fluorescence analysis.

The methyl-CpG binding domain (MBD) family proteins can specifically bind methylated DNA sequences and thereby mediate gene transcription. In this study, we used neutral capillary electrophoresis coupled with laser-induced fluorescence to investigate the interactions of DNA and MBD2b, a model MBD family protein with the highest affinity. For this purpose, we synthesized 13 double-stranded oligonucleotides of varying length (20 bp to 80 bp) and of varying methylation density. The sequences of these oligonucleotides were adapted from a frequently methylated promoter region of human p16(INK4a) gene. We demonstrate that multiple MBD2b proteins can bind to one DNA molecule with a DNA length-dependent binding stoichiometry. Each MBD2b protein can occupy 20 nucleotides in a bound DNA molecule regardless of the methylation status of DNA. By binding multiple MBD2b proteins (up to four protein molecules) to one dsDNA molecule (80 bp), methylated and unmethylated DNA were bound at similar percentages. Although the total amount of the DNA-MBD2b complexes increases with increasing DNA length for both unmethylated and methylated DNA, the DNA-MBD2b complexes of 1:1 display more than 10-fold higher affinity for methylated DNA (e.g., 40 bp DNA) accompanying a 20-fold lower dissociation rate constant. Hence, our study clarifies for the first time that the specificity of MBD2b to methylated DNA decreases as more MBD2b monomers binding to the same region of DNA. Additionally, this study opens a new venue to improve MBD protein-based assays for detecting DNA methylation.

Imaging of nonuniform motion of single DNA molecules reveals the kinetics of varying-field isotachophoresis.

The nonuniform motion of charged species in a varying electric field may provide unique separation and focusing power for chemical, biochemical, and nanoscale studies. We imaged in real time the nonuniform motion of single DNA molecules under varying-field isotachophoresis (ITP) conditions. From the trajectories of single molecules, we obtained the time- and position-dependent electric field strength (E) and revealed the behavior of adaption barriers within electro-osmotic flow (EOF)-driven and EOF-independent ITP. We found that the initial terminating electrolyte zone of constant E is split into two zones: a highly adapted high-E zone and a low-E zone of gradually adapting electric field. The formation of the two unique zones is associated with the rate-limiting mass transfer barrier in EOF-driven ITP. As a result of the unique E distribution, DNA molecules first slow to a stop and then rapidly move backward to the leading electrolyte/terminating electrolyte boundary. This provides a novel mechanism for selective focusing of target molecules in dilute solutions of large volume. We show that the ITP focusing can improve the detection of single DNA molecules (limit of detection = 4 × 10(-17) mol/L), which are stochastically distributed at extremely low concentrations. The ITP strategy focuses individual molecules into a small volume that is matched with the focal point of single-molecule imaging.

Ascorbic acid enhances Tet-mediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals.

DNA hydroxymethylation and its mediated DNA demethylation are critical for multiple cellular processes, for example, nuclear reprogramming, embryonic development, and many diseases. Here, we demonstrate that a vital nutrient ascorbic acid (AA), or vitamin C (Vc), can directly enhance the catalytic activity of Tet dioxygenases for the oxidation of 5-methylcytosine (5mC). As evidenced by changes in intrinsic fluorescence and catalytic activity of Tet2 protein caused by AA and its oxidation-resistant derivatives, we further show that AA can uniquely interact with the C-terminal catalytic domain of Tet enzymes, which probably promotes their folding and/or recycling of the cofactor Fe(2+). Other strong reducing chemicals do not have a similar effect. These results suggest that AA also acts as a cofactor of Tet enzymes. In mouse embryonic stem cells, AA significantly increases the levels of all 5mC oxidation products, particularly 5-formylcytosine and 5-carboxylcytosine (by more than an order of magnitude), leading to a global loss of 5mC (∼40%). In cells deleted of the Tet1 and Tet2 genes, AA alters neither 5mC oxidation nor the overall level of 5mC. The AA effects are however restored when Tet2 is re-expressed in the Tet-deficient cells. The enhancing effects of AA on 5mC oxidation and DNA demethylation are also observed in a mouse model deficient in AA synthesis. Our data establish a direct link among AA, Tet, and DNA methylation, thus revealing a role of AA in the regulation of DNA modifications.

Fluorescence anisotropy reduction of allosteric aptamer for sensitive and specific protein signaling.

Real time protein signaling in a complex medium may provide a promising way for high-throughput protein analysis, but it is largely unmet due to the challenge of signal transduction and the interferences of nonspecific binding and high background. Our recent work indicates that a fluorescent aptamer can display a protein binding-induced reduction of fluorescence anisotropy (FA) (Zhang, D.; Lu, M.; Wang, H. J. Am. Chem. Soc. 2011, 133, 9188-9191), which is exclusively different from a traditionally simplified concept hinting a molecular size increase-induced FA increase. Inspired by this unexpected observation, we describe a novel FA reduction approach for protein signaling. The feasibility of this approach is demonstrated through the assays of a blood protein human α-thrombin and an oncoprotein human platelet-derived growth factor B-chain (PDGF-BB) using two screened fluorescent aptamers, respectively. By the developed FA reduction method, the spiked human α-thrombin in diluted serum can be detected at the concentration as low as 250 pM. In contrast, in a traditional molecular size-dependent FA assay, the thrombin spiked in diluted serum cannot induce reliable FA change even at a 256-fold higher concentration (64 nM). The results clearly show that the FA reduction approach has a dramatically enhanced specificity against target protein and high sensitivity in complex medium and is applicable to the no-separation based detection of proteins in biological matrixes.

Specific and sensitive fluorescence anisotropy sensing of guanine-quadruplex structures via a photoinduced electron transfer mechanism.

Fluorescence anisotropy (FA) is a homogeneous, ratiometric, and real-time analytical technology. By selective labeling of a guanine (G)-quadruplex motif with tetramethylrhodamine (TMR), here, it is established that a large reduction in FA response can be specifically associated with the unfolding → folding transition of G-quadruplex structures. On the basis of fluorescence intensity, polarization and lifetime analysis, and molecular docking simulation, the mechanism was found to be that the labeled fluorophore (TMR) can intramolecularly interact with adjacent G bases in an unfolded G-quadruplex motif, which allows for the photoinduced electron transfer (PET) occurring between the fluorophore and G bases, leading to a short fluorescence lifetime. Upon the folding of the motif to form a stable G-quadruplex structure, the intramolecular interactions and the concomitant PET could be eliminated with an increased fluorescence lifetime, leading to a large reduction in the FA response. On the basis of this mechanism, a novel, specific, and sensitive FA approach was developed for the detection of biologically and functionally important G-quadruplex structures. The approach is examined and validated using one normal G-quadruplex motif, five mutants, and six small cations and is potentially applicable to the study of G-quadruplexes at single molecule level, ligand screening, profiling of highly ordered DNA nanostructures, and biosensing.

CircASXL1 Knockdown Restrains Hypoxia-Induced DDP Resistance and NSCLC Progression by Sponging miR-206.

BACKGROUND: Non-small cell lung carcinoma (NSCLC) is a primary prevalent type of cancer in people worldwide. Cisplatin (DDP) has been widely used to treat NSCLC; however, its curative effect was restrained under hypoxia. In this study, the effects of hypoxia treatment on DDP resistance and NSCLC progression and underneath mechanism were revealed. METHODS: The expression of circular RNA ASXL1 (circASXL1) and microRNA-206 (miR-206) in NSCLC tissues, cells and hypoxia-mediated NSCLC cells was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of proliferation, metastasis and apoptosis-related proteins, drug resistance-related protein and hypoxia-inducible factor-1alpha (HIF-1α) protein was detected by Western blot. The effects of circASXL1 knockdown on hypoxia-induced DDP resistance and NSCLC progression were revealed by cell counting kit-8 proliferation (CCK-8), cell colony formation, transwell and flow apoptosis assays. RNA immunoprecipitation (RIP) assay was performed to determine whether circASXL1 could form silence-inducing complexes with miRNA. The associated relationship between circASXL1 and miR-206 was predicted by circBank online database, and identified by RNA pull-down and dual-luciferase reporter assays. The effects between circASXL1 knockdown and miR-206 downregulation on tumor growth in vivo were investigated by in vivo tumor formation assay. RESULTS: CircASXL1 expression was dramatically upregulated, whereas miR-206 was significantly down-regulated in NSCLC tissues, cells and hypoxia-mediated NSCLC cells as compared to control groups. CircASXL1 knockdown reversed hypoxia-mediated promotion effects on DDP resistance, cell proliferation, migration, and invasion, and inhibition impact on cell apoptosis, whereas these effects were restored by miR-206 inhibitor. Additionally, circASXL1 was found to form silence-inducing complexes with miRNA and act as a sponge of miR-206. CircASXL1 silencing downregulated HIF-1α expression by controlling miR-206 expression. Furthermore, circASXL1 silencing repressed tumor growth in vivo by sponging miR-206. CONCLUSION: CircASXL1 knockdown inhibited DDP resistance, cell proliferation, migration and invasion, whereas induced cell apoptosis under hypoxia by associating with miR-206 in NSCLC. This study provides a new sight in treating NSCLC with DDP under hypoxia.

The essential elements for the noncovalent association of two DNA ends during NHEJ synapsis.

One of the most central questions about the repair of a double-strand DNA break (DSB) concerns how the two free DNA ends are brought together - a step called synapsis. Using single-molecule FRET (smFRET), we show here that both Ku plus XRCC4:DNA ligase IV are necessary and sufficient to achieve a flexible synapsis of blunt DNA ends, whereas either alone is not. Addition of XLF causes a transition to a close synaptic state, and maximum efficiency of close synapsis is achieved within 20 min. The promotion of close synapsis by XLF indicates a role that is independent of a filament structure, with action focused at the very ends of each duplex. DNA-PKcs is not required for the formation of either the flexible or close synaptic states. This model explains in biochemical terms the evolutionarily central synaptic role of Ku, X4L4, and XLF in NHEJ for all eukaryotes.

ATPase activity tightly regulates RecA nucleofilaments to promote homologous recombination.

Homologous recombination (HR), catalyzed in an evolutionarily conserved manner by active RecA/Rad51 nucleofilaments, maintains genomic integrity and promotes biological evolution and diversity. The structures of RecA/Rad51 nucleofilaments provide information critical for the entire HR process. By exploiting a unique capillary electrophoresis-laser-induced fluorescence polarization assay, we have discovered an active form of RecA nucleofilament, stimulated by ATP hydrolysis, that contains mainly unbound nucleotide sites. This finding was confirmed by a nuclease protection assay and electron microscopy (EM) imaging. We further found that these RecA-unsaturated filaments promote strand exchange in vitro and HR in vivo. RecA mutants (P67D and P67E), which only form RecA-unsaturated nucleofilaments, were able to mediate HR in vitro and in vivo, but mutants favoring the formation of the saturated nucleofilaments failed to support HR. We thus present a new model for RecA-mediated HR in which RecA utilizes its intrinsic DNA binding-dependent ATPase activity to remodel the nucleofilaments to a less saturated form and thereby promote HR.