protein-DNA Complexes Research Articles

MP79-14 LNCRNA-P21 ALTERS THE ANTIANDROGEN ENZALUTAMIDE-INDUCED PROSTATE CANCER NEUROENDOCRINE DIFFERENTIATION VIA MODULATING THE EZH2/STAT3 SIGNALING

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) IV (MP79)1 Apr 2020MP79-14 LNCRNA-P21 ALTERS THE ANTIANDROGEN ENZALUTAMIDE-INDUCED PROSTATE CANCER NEUROENDOCRINE DIFFERENTIATION VIA MODULATING THE EZH2/STAT3 SIGNALING Jie Luo*, Jialin Meng, Edward M. Messing, Jean V. Joseph, Shuyuan Yeh, and Chawnshang Chang Jie Luo*Jie Luo* More articles by this author , Jialin MengJialin Meng More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , Jean V. JosephJean V. Joseph More articles by this author , Shuyuan YehShuyuan Yeh More articles by this author , and Chawnshang ChangChawnshang Chang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000971.014AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancer (PCa) is the most prevalent male cancer in the U.S, and the current standard androgen deprivation therapy (ADT) with the FDA-approved antiandrogen Enzalutamide (Enz, also known as MDV3100) may effectively suppress the castration resistant PCa (CRPC) and extend CRPC patients’ survival an extra 4.8 months. However, ADT with Enz treatment (ADT-Enz) may also induce some unwanted adverse effects including development of Enz-resistance and increasing the PCa cell invasion as demonstrated in several preclinical models, as well as promoting the neuroendocrine (NE) differentiation (NED). METHODS: The viabilities of cells were determined by MTT (Sigma) assays and WST-1 assays (Cayman Chemical). The wildtype (wt) or mutant (mut) lncRNA-p21 promoters were constructed into PGL3-basic plasmid (Promega) to do the luciferase assays and luciferase activity was measured by Dual-Luciferase Assay reagent (Promega). Protein-DNA complexes were detected by Chromatin immunoprecipitation (ChIP) assays. In vivo mouse models were generated by PDX implantation and treated with different compound treatments. RESULTS: Results from cell lines and human clinical sample surveys revealed that lncRNA-p21 expression is up-regulated in NE PCa and Enz treatment could increase the lncRNA-p21 to induce the NED. Mechanism dissection revealed that Enz could promote the lncRNA-p21 transcription viaaltering the androgen receptor (AR) binding to different androgen-response-elements, which switch the EZH2 function from histone-methyltransferase to non-histone methyltransferase, consequently methylating the STAT3 to promote the NED. Preclinical studies using the PDX mouse model proved that EZH2 inhibitor could block the Enz-induced NED. CONCLUSIONS: In summary, our results suggest how Enz treatment can promote NED, and provide some potential therapies of using EZH2 inhibitors to target the AR/lncRNA-p21/EZH2/STAT3 axis to reduce the Enz-induced unwanted adverse effects of promoting the NED. Source of Funding: This work was supported by NIH grants (CA122840 and CA256700), and Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH102-TD-B-111-004) to China Medical University, Taiwan. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e1221-e1221 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jie Luo* More articles by this author Jialin Meng More articles by this author Edward M. Messing More articles by this author Jean V. Joseph More articles by this author Shuyuan Yeh More articles by this author Chawnshang Chang More articles by this author Expand All Advertisement PDF downloadLoading ...

A Protein in the Yeast Saccharomyces cerevisiae Presents DNA Binding Homology to the p53 Checkpoint Protein and Tumor Suppressor.

Saccharomyces cerevisiae does not contain a p53 homolog. Utilizing this yeast as an in vivo test tube model, our aim was to investigate if a yeast protein would show p53 DNA binding homology. Electrophoretic mobility shift analyses revealed the formation of specific DNA-protein complexes consisting of S. cerevisiae nuclear protein(s) and oligonucleotides containing p53 DNA binding sites. A S. cerevisiae p53 binding site factor (Scp53BSF) bound to a p53 synthetic DNA-consensus sequence (SCS) and a p53 binding-site sequence from the MDM2 oncogene. The complexes were of comparable size. Like mammalian p53, the affinity of Scp53BSF for the SCS oligonucleotide was higher than for the MDM2 oligonucleotide. Binding of Scp53BSF to the SCS and MDM2 oligonucleotides was strongly competed by unlabeled oligonucleotides containing mammalian p53 sites, but very little by a mutated site oligonucleotide. Importantly, Scp53BSF-DNA binding activity was significantly induced in extracts from cells with DNA damage. This resulted in dose-dependent coordinated activation of transcription when using p53-binding site reporter constructs. An ancient p53-like DNA binding protein may have been found, and activation of DNA-associated factors to p53 response elements may have functions not yet determined.

Tight DNA-protein complexes isolated from barley seedlings are rich in potential guanine quadruplex sequences.

BackgroundThe concept of chromatin domains attached to the nuclear matrix is being revisited, with nucleus described as a set of topologically associating domains. The significance of the tightly bound to DNA proteins (TBP), a protein group that remains attached to DNA after its deproteinization should be also revisited, as the existence of these interactions is in good agreement with the concept of the topologically associating domain. The work aimed to characterize the DNA component of TBP isolated from barley seedlings.MethodsThe tight DNA-protein complexes from the first leaves, coleoptiles, and roots of barley seedlings were isolated by purification with chromatography on nitrocellulose or exhaustive digestion of DNA with DNase I. Cloning and transformation were performed using pMOSBBlue Blunt Ended Cloning Kit. Inserts were amplified by PCR, and sequencing was performed on the MegaBace 1000 Sequencing System. The BLAST search was performed using sequence databases at NCBI, CR-EST, and TREP and Ensembl Plants databases. Comparison to MAR/SAR sequences was performed using http://smartdb.bioinf.med.uni-goettingen.de/cgi-bin/SMARtDB/smar.cgi database. The prediction of G quadruplexes (GQ) was performed with the aid of R-studio library pqsfinder. CD spectra were recorded on a Chirascan CS/3D spectrometer.ResultsAlthough the barley genome is AT-rich (43% of GC pairs), most DNA fragments associated with TBP were GC-rich (up to 70% in some fractions). Both fractionation procedures yielded a high proportion of CT-motif sequences presented predominantly by the 16-bp CC(TCTCCC)2 TC fragment present in clones derived from the TBP-bound DNA and absent in free DNA. BLAST analysis revealed alignment with different barley repeats. Some clones, however, aligned with both nuclear and chloroplast structural genes. Alignments with MAR/SAR motifs were very few. The analysis produced by the pqsfinder program revealed numerous potential quadruplex-forming sites in the TBP-bound sequences. A set of oligonucleotides containing sites of possible GQs were designed and ordered. Three of them represented the minus strand of the CT-repeat. Two were derived from sequences of two clones of nitrocellulose retained fraction from leaves and contained GC-rich motifs different from the CT motif. Circular dichroism spectroscopy revealed profound changes in spectra when oligonucleotides were incubated with 100 mM KCl. There was either an increase of positive band in the area of 260 nm or the formation of a positive band at 290 nm. In the former case, changes are typical for parallel G-quadruplexes and, in the latter, 3 + 1 structures. DiscussionThe G-quadruplexes anchor proteins are probably involved in the maintenance of the topologically associated domain structure.

Suppressing proteasome mediated processing of topoisomerase II DNA-protein complexes preserves genome integrity.

Topoisomerase II (TOP2) relieves topological stress in DNA by introducing double-strand breaks (DSBs) via a transient, covalently linked TOP2 DNA-protein intermediate, termed TOP2 cleavage complex (TOP2cc). TOP2ccs are normally rapidly reversible, but can be stabilized by TOP2 poisons, such as the chemotherapeutic agent etoposide (ETO). TOP2 poisons have shown significant variability in their therapeutic effectiveness across different cancers for reasons that remain to be determined. One potential explanation for the differential cellular response to these drugs is in the manner by which cells process TOP2ccs. Cells are thought to remove TOP2ccs primarily by proteolytic degradation followed by DNA DSB repair. Here, we show that proteasome-mediated repair of TOP2cc is highly error-prone. Pre-treating primary splenic mouse B-cells with proteasome inhibitors prevented the proteolytic processing of trapped TOP2ccs, suppressed the DNA damage response (DDR) and completely protected cells from ETO-induced genome instability, thereby preserving cellular viability. When degradation of TOP2cc was suppressed, the TOP2 enzyme uncoupled itself from the DNA following ETO washout, in an error-free manner. This suggests a potential mechanism of developing resistance to topoisomerase poisons by ensuring rapid TOP2cc reversal.

Abstract P3-11-12: Epigenetic modulation to deepen and prolong the response to PARP inhibitors

Abstract Background: BRCA1 and BRCA2 are key mediators of DNA damage repair response including the homologous recombination repair (HRR) pathway. Mutations in these genes predispose women to various cancers including breast, ovarian and pancreatic cancers. A new class of drugs, the poly (ADP-ribose) polymerase inhibitors (PARPi), were designed to leverage impaired DNA repair in BRCA-mutated tumors and are FDA approved for various cancers (breast, ovarian). Efforts to combine PARPi with cytotoxic agents show increased efficacy but overlapping toxicities hamper their tolerability. Further, the short duration of response to PARPi monotherapy in diseases like breast cancer has inspired the search for PARPi combinations with other agents including epigenetic modifiers. To overcome these hurdles, we explored synergistic interactions between PARPi and DNA methyltransferases inhibitors (DNMTi) such as decitabine, a cytidine analog. When incorporated in newly synthesized DNA strands during S phase, these agents will trigger a covalent protein-DNA complex formation causing cell cycle arrest and cell death. Material & Methods: BRCA wild-type (WT) and BRCA-mutated breast cancer cell lines were used for in vitro analyses: SUM149PT and HCC-3153 (BRCA1-mutated), MDA-MB-231 and BT-549 (BRCA-WT), UCRP231A (a CRISPR/Cas9 engineered BRCA2-mutated from parental MDA-MB-231), and MCF10A (a BRCA-WT non-tumorigenic breast cell line). The combined effect of talazoparib (PARPi) and decitabine (DNMTi) was analyzed using Combenefit® software to determine their synergistic interaction. Cell cycle analyses, dead/alive cells ratio analyses, as well as pH2AX levels were evaluated. In vivo analyses included xenograft models with SUM149PT as well as the parental BRCA-WT MDA-MB-231 and BRCA2-mutated MDA-MB-231 (UCRP231A) cells in immunodeficient NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ mice. Results: Our preclinical data in BRCA-deficient breast cancer cell lines, demonstrated a synergistic inhibition of cell growth and enhancement of cell death at concentrations of talazoparib and decitabine where each agent individually had minimal efficacy. In cells with intact HRR pathways, the drug combination showed an enhanced inhibition of cell growth but was not synergistic in inducing cell death compared to BRCA deficient cell lines. This difference was further manifested by different cell cycle profiles: non-BRCA mutated cells arrested in S-phase while BRCA-mutated cells progressed through S-phase without arrest and entered cell death from G2/M. In vivo mouse data showed a significant synergistic interaction between the two drugs without enhanced toxicity. Conclusion: The ability to induce a synergistic interaction between talazoparib and decitabine at low drug concentrations present a promising therapeutic strategy for patients with HRR-deficient tumors. Ongoing experiments are being conducted to generate additional engineered BRCA- and other HRR-deficient cell lines (ATM, CHEK2 and others) through CRISPR/Cas9 DNA editing system and further delineate the differential mechanism of interaction between the HRR-deficient and HRR-intact cell lines in preparation for clinical testing. Citation Format: Romain Pacaud, Mallika Dhawan, Scott Thomas, Elysia Roche, Jose Garcia, Nela Pawlowska, Pamela Munster. Epigenetic modulation to deepen and prolong the response to PARP inhibitors [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-12.

Overcharging of the Zinc Ion in the Structure of the Zinc-Finger Protein Is Needed for DNA Binding Stability

Zinc-finger structure, in which a Zn2+ ion binds to four cysteines or histidines in a tetrahedral structure, is a very common motif of nucleic acid-binding proteins. The corresponding interaction model is present in 3% of the genes in the human genome. As a result, the zinc finger has been extremely useful in various therapeutic and research capacities and in biotechnology. In a stable configuration of the zinc finger, the cysteine amino acids are deprotonated and become negatively charged. Thus, the Zn2+ ion is overscreened by four cysteine charges (overcharged). Whether this overcharged configuration is also stable when such a negatively charged zinc finger binds to a negatively charged DNA molecule is unknown. We investigated how the deprotonated state of cysteine influences its structure, dynamics, and function in binding to DNA molecules by using an all-atom molecular dynamics simulation up to the microsecond range of an androgen receptor protein dimer. Our results showed that the deprotonated state of cysteine residues is essential for the mechanical stabilization of the functional, folded conformation. This state stabilizes not only the protein structure but also the protein-DNA binding complex. The differences in the structural and energetic properties of the two sequence-identical monomers are also investigated and show the strong influence of DNA on the structure of the zinc-finger protein dimer upon complexation. Our result can potentially lead to a better molecular understanding of one of the most common classes of zinc fingers.

Multiple protein-DNA interfaces unravelled by evolutionary information, physico-chemical and geometrical properties.

Interactions between proteins and nucleic acids are at the heart of many essential biological processes. Despite increasing structural information about how these interactions may take place, our understanding of the usage made of protein surfaces by nucleic acids is still very limited. This is in part due to the inherent complexity associated to protein surface deformability and evolution. In this work, we present a method that contributes to decipher such complexity by predicting protein-DNA interfaces and characterizing their properties. It relies on three biologically and physically meaningful descriptors, namely evolutionary conservation, physico-chemical properties and surface geometry. We carefully assessed its performance on several hundreds of protein structures and compared it to several machine-learning state-of-the-art methods. Our approach achieves a higher sensitivity compared to the other methods, with a similar precision. Importantly, we show that it is able to unravel 'hidden' binding sites by applying it to unbound protein structures and to proteins binding to DNA via multiple sites and in different conformations. It is also applicable to the detection of RNA-binding sites, without significant loss of performance. This confirms that DNA and RNA-binding sites share similar properties. Our method is implemented as a fully automated tool, [Formula: see text], freely accessible at: http://www.lcqb.upmc.fr/JET2DNA. We also provide a new dataset of 187 protein-DNA complex structures, along with a subset of 82 associated unbound structures. The set represents the largest body of high-resolution crystallographic structures of protein-DNA complexes, use biological protein assemblies as DNA-binding units, and covers all major types of protein-DNA interactions. It is available at: http://www.lcqb.upmc.fr/PDNAbenchmarks.

Understanding Protein-DNA Recognition in the Context of Multi-Scale Genome Organization

Many structures of protein-DNA complexes have been solved and high-throughput binding assays were developed because structural biology and genomics researchers were equally puzzled by the question of how proteins bind DNA with high specificity. The understanding of protein-DNA binding has advanced through data generated by structural biology and genomics, and the combination of insights gained by both fields of research. Genomic data has become available for protein-DNA binding under cellular conditions. However, a mechanistic understanding of in vivo binding of transcription factors to DNA is still lacking. Different determinants for in vivo binding have been revealed but it is not clear to which extent each of those layers contributes to specificity [Slattery, et al. Absence of a simple code: how transcription factors read the genome. Trends Biochem. Sci. 39, 381-399 (2014)]. Moreover, these determinants play various roles for DNA binding of proteins from different families. Various layers of specificity relate to the genome organization in the nucleus at multiple scales. The factors discussed in this talk will include DNA methylation at the nucleotide scale [Rao, et al. Systematic prediction of DNA shape changes due to CpG methylation explains epigenetic effects on protein-DNA binding. Epigenetics Chromatin 11, 6 (2018)] and histone modifications at the nucleosome scale [Xin and Rohs. Relationship between histone modifications and transcription factor binding is protein family specific. Genome Res. 28, 321-333 (2018)]. The talk will also show how new computational such as deep learning can be used to study the impact of binding specificity determinants at multiple scales.

PreDBA: A heterogeneous ensemble approach for predicting protein-DNA binding affinity

The interaction between protein and DNA plays an essential function in various critical natural processes, like DNA replication, transcription, splicing, and repair. Studying the binding affinity of proteins to DNA helps to understand the recognition mechanism of protein-DNA complexes. Since there are still many limitations on the protein-DNA binding affinity data measured by experiments, accurate and reliable calculation methods are necessarily required. So we put forward a computational approach in this paper, called PreDBA, that can forecast protein-DNA binding affinity effectively by using heterogeneous ensemble models. One hundred protein-DNA complexes are manually collected from the related literature as a data set for protein-DNA binding affinity. Then, 52 sequence and structural features are obtained. Based on this, the correlation between these 52 characteristics and protein-DNA binding affinity is calculated. Furthermore, we found that the protein-DNA binding affinity is affected by the DNA molecule structure of the compound. We classify all protein-DNA compounds into five classifications based on the DNA structure related to the proteins that make up the protein-DNA complexes. In each group, a stacked heterogeneous ensemble model is constructed based on the obtained features. In the end, based on the binding affinity data set, we used the leave-one-out cross-validation to evaluate the proposed method comprehensively. In the five categories, the Pearson correlation coefficient values of our recommended method range from 0.735 to 0.926. We have demonstrated the advantages of the proposed method compared to other machine learning methods and currently existing protein-DNA binding affinity prediction approach.

Native Mass Spectrometry of Protein and DNA Complexes Prepared in Nonvolatile Buffers.

Inorganic salts and nonvolatile-buffer components affect the structure and stability of proteins, and some protein complexes are unable to maintain their function and structure without them. However, it is well-known that these components cause suppression of analyte ionization during the electrospray ionization process. Thus, to establish appropriate methods for observation of the intact ions of protein and DNA complexes by native mass spectrometry (native MS) in the presence of nonvolatile buffer components, we herein examined the effect of ammonium acetate addition to a model homotetramer protein, alcohol dehydrogenase (ADH), which was prepared in a range of nonvolatile buffers, including Tris-HCl, phosphate, and HEPES buffers. Furthermore, native MS of nucleosome core particle (NCP), a large protein-DNA complex, prepared in nonvolatile buffer, was also examined. Intact ADH and NCP ions could be observed upon the addition of ammonium acetate, but NCP does not require as high of a concentration of ammonium acetate as ADH. Well-resolved peaks with different charge numbers could be observed for NCP prepared in Tris-HCl by addition of a lower amount of ammonium acetate than for ADH. This suggests that the effects of additives on native MS of biomolecular complexes can vary depending on the intramolecular interactions present. More specifically, NCP is stabilized mainly by electrostatic interactions, whereas the ADH tetramer depends on the presence of hydrophobic interactions between the four subunits. The results presented herein therefore are expected to contribute to structural biology studies of unstable protein-DNA complexes that are formed transiently during the transcription process.

Model for DNA Interactions with Proteins and Other Large Ligands: Extracting Physical Chemistry from Pure Mechanical Measurements.

We present a new model to describe DNA interactions with large ligands such as proteins, based on a quenched-disorder equation for ligand binding along the double helix and on Manning's description for the local changes of the persistence length at the binding sites. Such a model allows one to extract the physical chemistry of the interactions from pure mechanical measurements, such as those typically performed with DNA-protein complexes in force spectroscopy assays. We have tested the proposed methodology here to investigate the DNA interaction with the protein lysozyme, determining binding parameters such as the equilibrium association constant, the cooperativity degree of the binding reaction, and the fraction of neutralized charges on the phosphate backbone. The model also allows one to get information on the size and positional conformation of the bound proteins.

NMR Observation of Intermolecular Hydrogen Bonds between Protein Tyrosine Side-Chain OH and DNA Phosphate Groups.

Hydrogen bonds between protein side-chain hydroxyl (OH) and phosphate groups are one of the most common types of intermolecular hydrogen bonds in protein-DNA/RNA complexes. Using NMR spectroscopy, we identified and characterized the hydrogen bonds between tyrosine side-chain OH and DNA phosphate groups in a protein-DNA complex. These OH groups exhibited relatively slow hydrogen-exchange rates and sizable scalar couplings between hydroxyl 1H and DNA phosphate 31P nuclei across the hydrogen bonds. Information about intermolecular hydrogen bonds facilitates investigations of the DNA/RNA recognition by the protein.

HMGA2 Regulates Circular RNA ASPH to Promote Malignant Phenotypes in Lung Adenocarcinoma

Background: The HMGA2 gene has been identified as an oncogene in many malignancies. The regulatory effects of HMGA2 on protein-coding oncogenes have been demonstrated by multiple studies. However, the effects of HMGA2 on circular RNAs (circRNAs) in malignancies remain unclear. Methods: CircRNA high-throughput sequencing analysis was performed on stable HMGA2-overexpressing A549 cells. qRT-PCR analysis in A549 cells and tissue samples was used to screen for the candidate circRNA. The effects of HMGA2 on circular RNA ASPH (circASPH) and its host gene were evaluated using RT-PCR. A chromatin immunoprecipitation assay was performed to detect DNA-protein complexes. Co-IP followed by mass spectrometry was used to determine the proteins interacting with HMGA2. The proliferation, migration, and invasion of lung adenocarcinoma cell lines were examined by EdU, wound scratch, and transwell invasion assays, respectively. The effect of circASPH on tumor growth in vivo was verified using a mouse xenograft assay. Luciferase, RNA FISH, and biotin-coupled miRNA capture assays were used to evaluate the interaction between circASPH and microRNAs. Findings: CircASPH expression levels were significantly higher in cancerous tissues. Patients with higher levels of circASPH had a shorter overall survival. CircASPH was found to be directly regulated by HMGA2 and Twist1. CircASPH promoted the malignant phenotypes of lung adenocarcinoma. CircASPH acted as a molecular sponge for miR-370 and abrogated miR-370-mediated inhibition of HMGA2. The oncogenic function of circASPH was HMGA2-dependent. Interpretation: These findings reveal the oncogenic functions of the HMGA2-circASPH-HMGA2 axis and may be useful in developing circRNA-based therapeutic strategies for lung adenocarcinoma. Funding Statement: The National Natural Science Foundation of China (grant 81702287 to Dr. Wei-Lin Liao and grant 81800443 to Dr. Ye Ma). Declaration of Interests: The authors stated: None. Ethics Approval Statement: All the study protocols were approved by the Institutional Review Boards of General Hospital of Western Theater Command. For examinations utilizing human lung tissue specimens, all patients gave written informed consent. The animal studies were performed in accordance with the institutional ethics guidelines for animal experiments.

Atomistic insight into sequence-directed DNA bending and minicircle formation propensity in the absence and presence of phased A-tracts.

Bending of double-stranded (ds) DNA plays a crucial role in many important biological processes and is relevant for nanotechnological applications. Among all the elements that have been studied in relation to dsDNA bending, A-tracts stand out as one of the most controversial. The "ApA wedge" theory was disproved when a series of linear polynucleotides containing phased 5'-A4T4-3' or 5'-T4A4-3' runs were shown to be bent or straight, respectively, and crystallographic evidence revealed that A-tracts are unbent. Furthermore, some of the smallest dsDNA minicircles described to date (~ 100bp in size) lack A-tracts and are subjected to varying levels of torsional stress. Representative DNA sequences from this experimental background were modeled in atomic detail and their dynamic behavior was simulated over hundreds of nanoseconds using the AMBER force field ParmBSC1. Subsequent analysis of the resulting trajectories allowed us to (i) unambiguously establish the location of the bends in all cases; (ii) identify the structural elements that are directly responsible for the macroscopically detected curvature; and (iii) reveal the importance not only of coherently summing the effects of the bending elements when they are in synchrony with the natural repeat of the helix (i.e. separated by an integral number of helical turns) but also when alternated with a half-integral separation of opposite effects. We conclude that the major determinant of the macroscopically observed bending is the proper grouping and phasing of the positive roll imposed by pyrimidine-purine (YR) steps and the negative or null roll characteristic of RY steps and A-tracts, respectively. This conclusion is in very good agreement with the structural parameters experimentally derived for much smaller DNA molecules either alone or as found in DNA-protein complexes. We expect that this work will pave the way for future studies on drug-induced DNA bending, DNA shape readout by transcription factors, structure of circular extrachromosomal DNA, and custom design of curved DNA origami scaffolds.

Telomere uncapping by common oxidative guanine lesions: Insights from atomistic models

Oxidative damage to DNA is widely known to contribute to aging and disease. This relationship has been extensively studied for telomeres – structures that cap chromosome ends – due to their role in cell proliferation and senescence, and exceptional susceptibility to oxidation. Indeed, the repetitive telomeric DNA sequence contains the 5′-GGG-3′ motif that has the lowest ionization potential of all trinucleotides. Accordingly, experiments consistently show that telomeric oxidative lesions are more abundant and persistent than elsewhere in the genome. This led to a hypothesis that telomeres act as sensors of prolonged oxidative stress and prevent carcinogenesis, as disruption of telomeric integrity triggers senescence or apoptosis. Here, we use atomistic alchemical Molecular Dynamics simulations to perform a combinatorial assessment of changes in DNA binding affinity of telomeric proteins induced by oxidative guanine lesions. We rank lesions by their effect on telomere integrity, as well as telomeric proteins by their sensitivity to DNA oxidation. While the binding of most proteins is abolished by DNA oxidation, HOT1 emerges as a notable exception, suggesting its potential role in sensing of oxidative damage. Through statistical analysis and free energy decomposition, we also identify common trends in structural responses of protein-DNA complexes that contribute to decreased binding affinity.

The effect of obstacles in multi-site protein target search with DNA looping

Many fundamental biological processes are regulated by protein-DNA complexes called synaptosomes, which possess multiple interaction sites. Despite the critical importance of synaptosomes, the mechanisms of their formation are not well understood. Because of the multisite nature of participating proteins, it is widely believed that their search for specific sites on DNA involves the formation and breaking of DNA loops and sliding in the looped configurations. In reality, DNA in live cells is densely covered by other biological molecules that might interfere with the formation of synaptosomes. In this work, we developed a theoretical approach to evaluate the role of obstacles in the target search of multisite proteins when the formation of DNA loops and the sliding in looped configurations are possible. Our theoretical method is based on analysis of a discrete-state stochastic model that uses a master equations approach and extensive computer simulations. It is found that the obstacle slows down the search dynamics in the system when DNA loops are long-lived, but the effect is minimal for short-lived DNA loops. In addition, the relative positions of the target and the obstacle strongly influence the target search kinetics. Furthermore, the presence of the obstacle might increase the noise in the system. These observations are discussed using physical-chemical arguments. Our theoretical approach clarifies the molecular mechanisms of formation of protein-DNA complexes with multiple interactions sites.

Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes.

DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional footprinting protocols, because it provides a method to isolate the protein-DNA complex from a native gel after treatment with the footprinting agent, thus removing the bound DNA from the free DNA or other protein-DNA complexes. The DNA is then extracted from the isolated complex before electrophoresis on a sequencing gel to determine the footprinting pattern. This analysis provides a possible solution for those who have been unable to use traditional footprinting methods to determine protein-DNA contacts.

Novel Protein-oligonucleotide Conjugation Method Involving a High-affinity Capture HaloTag.

Highly sensitive quantitative protein profiling can play a key role in the early diagnosis of diseases, such as autoimmune diseases and cancer. We developed a modified protein-oligonucleotide conjugation method termed HaloTag-mediated barcoding, for quantifying protein molecules at a higher sensitivity than conventional protein quantification methods. This novel and efficient conjugation method can be used to prepare HaloTag-barcoded proteins using a click chemistry-based labeling technique. Here, we describe the preparation of protein-DNA complexes and detection of protein-protein interactions which can be used in a HaloTag protein barcode assay to detect an antibody. The protocol includes procedures for preparing the ligand-oligonucleotide complex, plasmid DNA preparation for protein expression, and preparation of the protein-oligonucleotide complex. The described click reaction-based protocols simplify the conventional amine-ester reaction methods which require additional steps for chromatography purification.

The effect of carbon nanotube on the structure of H–NS protein DNA complex: molecular dynamics approach

The effect of carbon nanotube on the structure of H–NS protein DNA complex: molecular dynamics approach

Programmable Assembly of DNA-protein Hybrid Structures

DNA is the genetic information carrier for most known living organisms on Earth, while proteins are the functional component that carry out most biological processes. Many natural machineries are DNA-protein hybrid complexes to cooperatively and efficiently conduct sophisticated biological tasks. It has drawn increasing interest to the research field to construct artificial DNA-protein hybrid structures towards a variety of applications including biological studies, nanofabrication, biomedical research, etc. In this regard, here in this report we reviewed the up-to-date progress on making DNA-protein hybrid structures, with a particular focus on DNA nanotechnology-enabled programmable assembly of DNA-protein hybrid structures.