Local DNA Research Articles

Attenuated Expression of SLCO2A1 Caused by DNA Methylation in Pediatric Inflammatory Bowel Disease.

SLCO2A1 encodes a prostaglandin (PG) transporter, and autosomal recessive pathogenic variants of this gene cause chronic enteropathy associated with SLCO2A1. It is unclear whether a heterozygous pathogenic variant of SLCO2A1 has a role in the pathogenesis of other types of inflammatory bowel disease (IBD). In this study, we investigated the possible involvement of a local epigenetic alteration in SLCO2A1 in patients with a heterozygous pathogenic variant. We conducted whole-exome sequencing of samples from 2 sisters with suspected monogenic IBD. In addition, we performed bisulfite sequencing using DNA extracted from their small and large intestine samples to explore epigenetic alterations. A heterozygous splicing site variant, SLCO2A1:c.940 + 1G > A, was detected in both patients. To explore the possible involvement of epigenetic alterations, we analyzed protein and messenger RNA expression of SLCO2A1, and observed attenuated SLCO2A1 expression in the inflamed lesions of these patients compared with that in the control individuals. Furthermore, bisulfite sequencing indicated dense methylation in the promoter region of SLCO2A1 only in the inflamed lesions of both patients. The urinary PG metabolite levels in these patients were comparable to those in patients with chronic enteropathy associated with SLCO2A1 and higher than those in the control individuals. We found considerably higher levels of the metabolites in patient 1, who showed more severe symptoms than patient 2. Local DNA methylation attenuated SLCO2A1 expression, which may evoke local inflammation of the mucosa by the unincorporated PG. These findings may improve our understanding of the epigenetic mechanisms underlying IBD development.

RegCloser: a robust regression approach to closing genome gaps

BackgroundClosing gaps in draft genomes leads to more complete and continuous genome assemblies. The ubiquitous genomic repeats are challenges to the existing gap-closing methods, based on either the k-mer representation by the de Bruijn graph or the overlap-layout-consensus paradigm. Besides, chimeric reads will cause erroneous k-mers in the former and false overlaps of reads in the latter.ResultsWe propose a novel local assembly approach to gap closing, called RegCloser. It represents read coordinates and their overlaps respectively by parameters and observations in a linear regression model. The optimal overlap is searched only in the restricted range consistent with insert sizes. Under this linear regression framework, the local DNA assembly becomes a robust parameter estimation problem. We solved the problem by a customized robust regression procedure that resists the influence of false overlaps by optimizing a convex global Huber loss function. The global optimum is obtained by iteratively solving the sparse system of linear equations. On both simulated and real datasets, RegCloser outperformed other popular methods in accurately resolving the copy number of tandem repeats, and achieved superior completeness and contiguity. Applying RegCloser to a plateau zokor draft genome that had been improved by long reads further increased contig N50 to 3-fold long. We also tested the robust regression approach on layout generation of long reads.ConclusionsRegCloser is a competitive gap-closing tool. The software is available at https://github.com/csh3/RegCloser. The robust regression approach has a prospect to be incorporated into the layout module of long read assemblers.

Mutation-site localized non-B DNA burden and survival heterogeneity in early-stage pancreatic cancer.

4166 Background: Pancreatic cancer is a highly aggressive disease with a poor prognosis, and one in which most measure of genomic instability (e.g., tumor mutation burden (TMB), fraction of genome altered (FGA)) have thus far, not proven informative in differentiating survival. Non-B DNA are alternative DNA forms that deviate from the canonical B-DNA structure with potential to increase susceptibility to mutations, leading to the development of cancer. The role of non-B in pancreatic cancer has not been fully explored. Herein, we investigate the relationship between gene mutation sites co-localized with non-B DNA motifs in terms of survival and B-DNA features. Methods: Using TCGA data, we derived a genome-wide mutation signature on 104 early-stage pancreatic patients. We introduced a metric, mutation-localized non-B burden (MLNB) by the number of non-b motifs containing mutation sites, normalized to the number of mutations and motif library size. We applied MLNB to patient-specific mutation signatures to derive an MLNB burden for non-B DNA structure motifs: G-quadruplexes (G4), Z-DNA, inverted repeats (IR), mirror repeats (MR), direct repeats (DR), and short tandem repeats (STR). We performed a cluster analysis on MLNB and compared groups based on overall survival (OS) in months (mos) using a log-rank test. Comparisons of B-DNA-derived molecular features among clusters were performed using a Kruskal-Wallis and Fisher Exact test. Results: Among the 104 early-stage pancreatic patients with a mutation signature, MLNB clustering resulted in six patient clusters that differentiated by non-B DNA structure, with DR burden the longest OS cluster (n=23, median OS=30mos), significantly (p < 0.05) differing as compared to IR (n=23, median OS=15 mos), STR (n=20, median OS=16mos), MR with lack of IR (n=14, median OS=13mos) and MR with IR (n=16, median OS=8mos). A mix of Z-DNA and G4 burden defined a cluster with shorter OS (n=22, median OS=14mos) as compared to DR, though not significant. Patients with the longest OS, MLNB-DR burden cluster had mutation signatures enriched in MAPK and Notch signaling pathways, as compared to the other clusters enriched with double-stranded break and mismatch repair (IR), hedgehog and WNT signaling (STR), and interleukin-4 signaling (MR) pathways. Among all clusters, CDKN2A deletion was most prevalent except for MLNB-DR burden cluster. No Significant differences were found between the MLNB clusters with age, race gender, KRAS and TP53 mutations, FGA, TMB, tumor purity, Bailey and Moffit subtypes. Conclusions: Our results point towards the potential use of MLNB as an emerging marker of site-structure genomic instability. Considering that triplex DNA is emerging as a marker genomic instability, and that we distinguish between two MR groups based on IR burden, our results may provide insights into various instability mechanisms associated with poor survival in pancreatic cancer.

Circulating tumour DNA concentration and genetic classification improve risk stratification in newly diagnosed patients with Diffuse Large B‐Cell Lymphoma

Introduction: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease with poor outcomes for the 40% of patients who relapse or are refractory to upfront therapy, and current prognostic tools are unable to identify many of those patients. Recently, novel prognostic biomarkers like genetic classification and circulating tumor DNA (ctDNA) have been developed. Here we explore the value these biomarkers to improve prognostic stratification in DLBCL. Methods: DNA was extracted from 2 to 5 ml of plasma and formalin-fixed paraffin-embedded (FFPE) tumor tissue obtained at diagnosis from patients with DLBCL treated with R-CHOP-like regimes. Samples were sequenced in an Illumia NovaSeq using a panel of 112 genes that are recurrently mutated in lymphoid neoplasms, and variants were called using a pipeline that follows GATK best practices. Genetic subtype (GS) was determined using LymphGen tool, excluding A53, as copy number variation data was unavailable. ST2, EZB MYC negative and BN2 subtypes were considered favorable, while N1, MCD, EZB MYC positive and other were considered unfavorable. ctDNA levels were reported as haploid genome equivalents per mL of plasma and expressed as a base 10 logarithm (log hGE/mL) Results: We included 46 patients with median age of 64.8 years, 58% advanced stage (III-IV) and 65% germinal center DLBCL by Hans’s algorithm. Somatic mutations were detected in FFPE in all patients and in ctDNA in 40 (87%) patients. Most frequently mutated genes were KMT2D, CREBBP, TP53, ARID1A, MYD88 and CARD11. The sensitivity of ctDNA to detect mutations present in paired FFPE samples was 66% (for all variants) and 74.5% (for variants with >5% allelic frequency in FFPE samples). 67.4% of patients were successfully classified by LymphGen, with a concordance between ctDNA and FFPE of 84.7%. Patients without detectable ctDNA presented localized disease (Ann Arbor stage I) and low DNA concentration in plasma (<15 ng/ml). In our cohort, 24 patients presented a poor R-IPI score with a 3-year progression-free survival (PFS) of 61.5% (Figure 1A). These patients were further stratified by ctDNA levels and GS, and those unfavorable GS or ctDNA concentration >4 log hGE/ml presented worse PFS and OS than without risk factors a 3-year PFS of 34.6% and 90.9%, respectively (Figure 1B). Patients were classified into three groups according IPI score modified by ctDNA levels (Figure 1C), with a 3-year PFS of 94.7%, 81.2% and 18.8%, and a 3-year overall survival (OS) of 100%, 87.5% and 37.5%, respectively (Figure 1D–E). Keywords: Diagnostic and Prognostic Biomarkers, Genomics, Epigenomics, and Other -Omics No conflicts of interests pertinent to the abstract.

Profiling the immune epigenome across global cattle breeds

BackgroundUnderstanding the variation between well and poorly adapted cattle breeds to local environments and pathogens is essential for breeding cattle with improved climate and disease-resistant phenotypes. Although considerable progress has been made towards identifying genetic differences between breeds, variation at the epigenetic and chromatin levels remains poorly characterized. Here, we generate, sequence and analyse over 150 libraries at base-pair resolution to explore the dynamics of DNA methylation and chromatin accessibility of the bovine immune system across three distinct cattle lineages.ResultsWe find extensive epigenetic divergence between the taurine and indicine cattle breeds across immune cell types, which is linked to the levels of local DNA sequence divergence between the two cattle sub-species. The unique cell type profiles enable the deconvolution of complex cellular mixtures using digital cytometry approaches. Finally, we show distinct sub-categories of CpG islands based on their chromatin and methylation profiles that discriminate between classes of distal and gene proximal islands linked to discrete transcriptional states.ConclusionsOur study provides a comprehensive resource of DNA methylation, chromatin accessibility and RNA expression profiles of three diverse cattle populations. The findings have important implications, from understanding how genetic editing across breeds, and consequently regulatory backgrounds, may have distinct impacts to designing effective cattle epigenome-wide association studies in non-European breeds.

FRET Imaging of Nonuniformly Distributed DNA SAMs on Gold Reveals the Role Played by the Donor/Acceptor Ratio and the Local Environment in Measuring the Rate of Hybridization.

Mixed DNA SAMs labeled with a fluorophore (either AlexaFluor488 or AlexaFluor647) were prepared on a single crystal gold bead electrode using potential-assisted thiol exchange and studied using Förster resonance energy transfer (FRET). A measure of the local environment of the DNA SAM (e.g., crowding) was possible using FRET imaging on these surfaces since electrodes prepared this way have a range of surface densities (ΓDNA). The FRET signal was strongly dependent on ΓDNA and on the ratio of AlexaFluor488 to AlexaFluor647 used to make the DNA SAM, which were consistent with a model of FRET in 2D systems. FRET was shown to provide a direct measure of the local DNA SAM arrangement on each crystallographic region of interest providing a direct assessment of the probe environment and its influence on the rate of hybridization. The kinetics of duplex formation for these DNA SAMs was also studied using FRET imaging over a range of coverages and DNA SAM compositions. Hybridization of the surface-bound DNA increased the average distance between the fluorophore label and the gold electrode surface and decreased the distance between the donor (D) and acceptor (A), both of which result in an increase in FRET intensity. This increase in FRET was modeled using a second order Langmuir adsorption rate equation, reflecting the fact that both D and A labeled DNA are required to become hybridized to observe a FRET signal. The self-consistent analysis of the hybridization rates on low and high coverage regions on the same electrode showed that the low coverage regions achieved full hybridization 5× faster than the higher coverage regions, approaching rates typically found in solution. The relative increase in FRET intensity from each region of interest was controlled by manipulating the donor to acceptor composition of the DNA SAM without changing the rate of hybridization. The FRET response can be optimized by controlling the coverage and the composition of the DNA SAM sensor surface and could be further improved with the use of a FRET pair with a larger (e.g., > 5 nm) Förster radius.

High-LET targeted microbeam irradiation induces local chromatin reorganization in living cells showing active basal mechanisms at highly complex DNA damage sites

DNA repair eukaryotic cells have additional protective mechanisms that avoid uncontrolled interaction of different parts of the chromatin and damaged regions. Key factors here are the regulation of chromatin density and mobility. The 4D (temporal and spatial) organization of chromatin is controlling this security barrier by regulating the accessibility of genes, flexibility of DNA, and its ability to move inside the nucleus. How this regulation mechanisms are involved in DNA repair upon radiation damage is until now rarely known but an important part to understand the enhanced effectiveness of high linear energy transfer (LET) particles. The damage recognition via PARP1 and the subsequent chromatin decondensation via PARylation is a crucial step in the DNA damage response (DDR). Upon We used the SNAKE microbeam with a beam spot size of &lt;1 µm to induce highly localized DNA damage in living cells using 55 MeV Carbon ions to investigate the chromatin rearrangements in the early stage of DDR. The nuclei were irradiated with a cross pattern consisting of 1000 ions per spot and 25 spots per cell either with one (11 000 Gy), two (22 000 Gy), or three crosses (33 000 Gy). The chromatin rearrangement was imaged live for several minutes after irradiation at the beam using SiR chromatin stain. Upon 91% of the cells show a localized decondensation starting from a few seconds up to minutes after irradiation. The chromatin is decondensed by 6%-8% in the beam path with a local condensation at the edges of up to 8%. Our results suggest that chromatin decondensation is a fast process in the first few seconds after damage induction. Furthermore, decondensation status does not change over minutes, which gives evidence that this process and therefore DDR is paused or even stopped. In combination with the existing knowledge about early reactions to damage induction our data support the model of PARP induced chromatin decondensation. Furthermore, it is evident that also ultra-high doses of radiation are, in first place not able to inactivate initial basal mechanisms as response to damage induction.

Combination of local immunogenic cell death-inducing chemotherapy and DNA vaccine increases the survival of glioblastoma-bearing mice

Immunotherapy efficacy as monotherapy is negligible for glioblastoma (GBM). We hypothesized that combining therapeutic vaccination using a plasmid encoding an epitope derived from GBM-associated antigen (pTOP) with local delivery of immunogenic chemotherapy using mitoxantrone-loaded PEGylated PLGA-based nanoparticles (NP-MTX) would improve the survival of GBM-bearing mice by stimulating an antitumor immune response. We first proved that MTX retained its ability to induce cytotoxicity and immunogenic cell death of GBM cells after encapsulation. Intratumoral delivery of MTX or NP-MTX increased the frequency of IFN-γ-secreting CD8 T cells. NP-MTX mixed with free MTX in combination with pTOP DNA vaccine increased the median survival of GL261-bearing mice and increased M1-like macrophages in the brain. The addition of CpG to this combination abolished the survival benefit but led to increased M1 to M2 macrophage ratio and IFN-γ-secreting CD4 T cell frequency. These results highlight the benefits of combination strategies to potentiate immunotherapy and improve GBM outcome.

The effects of RNA.DNA-DNA triple helices on nucleosome structures and dynamics

Noncoding RNAs (ncRNAs) are an emerging epigenetic factor and have been recognized as playing a key role in many gene expression pathways. Structurally, binding of ncRNAs to isolated DNA is strongly dependent on sequence complementary and results in the formation of an RNA.DNA-DNA (RDD) triple helix. However, in vivo DNA is not isolated but is rather packed in chromatin fibers, the fundamental unit of which is the nucleosome. Biochemical experiments have shown that ncRNA binding to nucleosomal DNA is elevated at DNA entry and exit sites and is dependent on the presence of the H3 N-terminal tails. However, the structural and dynamical bases for these mechanisms remain unknown. Here, we have examined the mechanisms and effects of RDD formation in the context of the nucleosome using a series of all-atom molecular dynamics simulations. Results highlight the importance of DNA sequence on complex stability, elucidate the effects of the H3 tails on RDD structures, show how RDD formation impacts the structure and dynamics of the H3 tails, and show how RNA alters the local and global DNA double-helical structure. Together, our results suggest ncRNAs can modify nucleosome, and potentially higher-order chromatin, structures and dynamics as a means of exerting epigenetic control.

Temporal recruitment of base excision DNA repair factors in living cells in response to different micro-irradiation DNA damage protocols

Laser micro-irradiation across the nucleus rapidly generates localized chromatin-associated DNA lesions permitting analysis of repair protein recruitment in living cells. Recruitment of three fluorescently-tagged base excision repair factors [DNA polymerase β (pol β), XRCC1 and PARP1], known to interact with one another, was compared in gene-deleted mouse embryonic fibroblasts and in those expressing the endogenous factor. A low energy micro-irradiation (LEMI) forming direct single-strand breaks and a moderate energy (MEMI) protocol that additionally creates oxidized bases were compared. Quantitative characterization of repair factor recruitment and sensitivity to clinical PARP inhibitors (PARPi) was dependent on the micro-irradiation protocol. PARP1 recruitment was biphasic and generally occurred prior to pol β and XRCC1. After LEMI, but not after MEMI, pol β and XRCC1 recruitment was abolished by the PARPi veliparib. Consistent with this, pol β and XRCC1 recruitment following LEMI was considerably slower in PARP1-deficient cells. Surprisingly, the recruitment half-times and amplitudes for pol β were less affected by PARPi than were XRCC1 after MEMI suggesting there is a XRCC1-independent component for pol β recruitment. After LEMI, but not MEMI, pol β dissociation was more rapid than that of XRCC1. Unexpectedly, PARP1 dissociation was slowed in the absence of XRCC1 as well with a PARPi after LEMI but not MEMI, suggesting that XRCC1 facilitates PARP1 dissociation from specific DNA lesions. XRCC1-deficient cells showed pronounced hypersensitivity to the PARPi talazoparib correlating with its known cytotoxic PARP1 trapping activity. In contrast to DNA methylating agents, PARPi only minimally sensitized pol β and XRCC1-deficient cells to oxidative DNA damage suggesting differential binding of PARP1 to alternate repair intermediates. In summary, pol β, XRCC1, and PARP1 display recruitment kinetics that exhibit correlated and unique properties that depend on the DNA lesion and PARP activity revealing that there are multiple avenues utilized in the repair of chromatin-associated DNA.

DNA methylation abnormalities induced by advanced maternal age in villi prime a high-risk state for spontaneous abortion

BackgroundAdvanced maternal age (AMA) has increased in many high-income countries in recent decades. AMA is generally associated with a higher risk of various pregnancy complications, and the underlying molecular mechanisms are largely unknown. In the current study, we profiled the DNA methylome of 24 human chorionic villi samples (CVSs) from early pregnancies in AMA and young maternal age (YMA), 11 CVSs from early spontaneous abortion (SA) cases using reduced representation bisulfite sequencing (RRBS), and the transcriptome of 10 CVSs from AMA and YMA pregnancies with mRNA sequencing(mRNA-seq). Single-cell villous transcriptional atlas presented expression patterns of targeted AMA-/SA-related genes. Trophoblast cellular impairment was investigated through the knockdown of GNE expression in HTR8-S/Vneo cells.ResultsAMA-induced local DNA methylation changes, defined as AMA-related differentially methylated regions (DMRs), may be derived from the abnormal expression of genes involved in DNA demethylation, such as GADD45B. These DNA methylation changes were significantly enriched in the processes involved in NOTCH signaling and extracellular matrix organization and were reflected in the transcriptional alterations in the corresponding biological processes and specific genes. Furthermore, the DNA methylation level of special AMA-related DMRs not only significantly changed in AMA but also showed more excessive defects in CVS from spontaneous abortion (SA), including four AMA-related DMRs whose nearby genes overlapped with AMA-related differentially expressed genes (DEGs) (CDK11A, C19orf71, COL5A1, and GNE). The decreased DNA methylation level of DMR near GNE was positively correlated with the downregulated expression of GNE in AMA. Single-cell atlas further revealed comparatively high expression of GNE in the trophoblast lineage, and knockdown of GNE in HTR8-S/Vneo cells significantly impaired cellular proliferation and migration.ConclusionOur study provides valuable resources for investigating AMA-induced epigenetic abnormalities and provides new insights for explaining the increased risks of pregnancy complications in AMA pregnancies.Graphical

Precise programming of multigene expression stoichiometry in mammalian cells by a modular and programmable transcriptional system

Context-dependency of mammalian transcriptional elements has hindered the quantitative investigation of multigene expression stoichiometry and its biological functions. Here, we describe a host- and local DNA context-independent transcription system to gradually fine-tune single and multiple gene expression with predictable stoichiometries. The mammalian transcription system is composed of a library of modular and programmable promoters from bacteriophage and its cognate RNA polymerase (RNAP) fused to a capping enzyme. The relative expression of single genes is quantitatively determined by the relative binding affinity of the RNAP to the promoters, while multigene expression stoichiometry is predicted by a simple biochemical model with resource competition. We use these programmable and modular promoters to predictably tune the expression of three components of an influenza A virus-like particle (VLP). Optimized stoichiometry leads to a 2-fold yield of intact VLP complexes. The host-independent orthogonal transcription system provides a platform for dose-dependent control of multiple protein expression which may be applied for advanced vaccine engineering, cell-fate programming and other therapeutic applications.

Sequence and chromatin features guide DNA double-strand break resection initiation

DNA double-strand breaks (DSBs) are cytotoxic genome lesions that must be accurately and efficiently repaired to ensure genome integrity. In yeast, the Mre11-Rad50-Xrs2 (MRX) complex nicks 5'-terminated DSB ends to initiate nucleolytic processing of DSBs for repair by homologous recombination. How MRX-DNA interactions support 5' strand-specific nicking and how nicking is influenced by the chromatin context have remained elusive. Using a deep sequencing-based assay, we mapped MRX nicks at single-nucleotide resolution next to multiple DSBs in the yeast genome. We observed that the DNA end-binding Ku70-Ku80 complex directed DSB-proximal nicks and that repetitive MRX cleavage extended the length of resection tracts. We identified a sequence motif and a DNA meltability profile that is preferentially nicked by MRX. Furthermore, we found that nucleosomes as well as transcription impeded MRX incisions. Our findings suggest that local DNA sequence and chromatin features shape the activity of this central DSB repair complex.

Tracing cellular interaction of circRNA-miRNA axis with Cu metal-organic framework supported DNA cascade assembly

Circular RNAs (circRNAs) can act as molecular sponges of microRNA (miRNA) to form circRNA-miRNA axis, which regulates the expressions of downstream proteins. Although the mechanism has been widely reported in various bioprocesses, there is still a lack of reliable and facile way to intuitively monitor and locate the interaction between circRNA and miRNA inside living cells. In this study, multiple DNA probes are designed and loaded onto two-dimensional Cu metal-organic framework (2D Cu-MOF) nanosheets for one-step analysis of circRNA-miRNA axis. The nanosheets serve as not only powerful fluorescence quenchers but also excellent nanocarriers of abundant DNA probes for further assembly. The Probes@Cu-MOF complex can be applied to track the circRNA-miRNA axis in living cells with high sensitivity and co-localization analysis. This platform combines the transmembrane advantage of nanosheets and the signal amplification ability of localized DNA cascade assembly, so it holds great potential for understanding the biological functions of circRNA-miRNAs in cancer pathogenesis and for therapeutic applications.

Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy.

Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigate Staphylococcus aureus Cas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single-guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets.

Evolutionary Trend in Passiflora Species Revealed by RAPD-PCR, Gene Specific ITS and rbcl Markers

Background: The present study represents a preliminary analysis of genetic diversity among Passiflora species using amplified genotypic data of specific ITS and rbcL sequences and non-specific RAPD-PCR markers for investigation of the molecular phylogeny.&#x0D; Methods: The PCR-RAPD uses ten primers for polymorphic DNA, which are compiled on NTSYS software to construct dendrogram. The gene specific ITS and rbcL primers are used for specific amplification from genomic. The amplified ITS and rbcL markers assembled using Maximum Parsimony (MP) and Maximum likelihood (ML) methods. The BLAST, CLUSTAL W, and MEGA 6.0 have been used to conclude final genetic relation tree.&#x0D; Results: The PCR-RAPD primers translate 133 random amplified polymorphic DNA. NTSYS dendrogram placed P. vitifolia from Ramdaspeth and Shankar Nagar Nagpur, India in same clade (similarity coefficient 0.609) confirming same origin Nagpur India. Moreover, P. foetida from England is not coming in same clade with Indian P. foetida showing geographically intra-specific variation. In addition, the change in a constructed tree was observed with respect to change in phylogeny methods MS/ML. The ITS MP consensus tree is supported by strong 100 bootstrap value, clusters P. vitifolia (HNI and RNI) and P. foetida (UAI and UGI) in equivalent clade. However, no single species have been recovered using rbcL in MP and ML method. Thus, it is inference that rbcL have tendency to differentiate Passiflora species not allowing clustering around same species in same clade and the ITS region having parsimony informative sites that provide valid resolution and identification at inter-intra species level.&#x0D; Conclusions: The evaluation of properties of RAPD indicates 100% PCR success, sometimes with low rate of amplification. The ITS region found to be best for identification at inter-intra species, On the contrary, rbcL region is good to distinguished inter species, making it best as local DNA barcode for marking a Passiflora species in phylogenetic community.

Investigating local DNA conformational trapping dynamics during DNA polymerase holoenzyme assembly and exonuclease proof-reading activity.

The sugar-phosphate backbones of DNA continuously undergo thermally-activated conformational fluctuations at equilibrium, which directly affect base-pair unstacking and hydrogen bond breaking. The influence of these motions on biomolecular function are particularly important at single-stranded (ss)—double-stranded (ds) DNA forks and primer-template (p/t) junctions. At these junctions, non-canonical structures that deviate from the Watson-Crick B-form DNA duplex may serve as recognition sites for protein binding, and the kinetics of these interactions likely play a central regulatory role for fundamental biological processes such as DNA replication and repair. The DNA polymerase/sliding clamp holoenzyme is the central protein complex that carries out the process of complementary nucleotide addition to nascent ssDNA template strands initiated at p/t junctions. In addition to playing a role in protein assembly, local DNA conformations resulting from nucleotide misincorporation regulate the exonuclease proof-reading activity of DNA polymerase. To study the details of these processive local DNA conformational dynamics, our group has developed a molecular probing method that relies on the rigid insertion of two fluorescent cyanine (Cy3) probes at opposite positions within the sugar-phosphate backbones of complementary DNA strands. Electrostatic coupling between probes results in delocalized optical properties that sensitively depend on relative probe orientations. Utilizing a series of complementary ensemble-based (i.e., fluorescence, absorbance, circular dichroism) and single-molecule spectroscopic methods, we study the local DNA backbone conformational dynamics as they relate to protein binding and activity. In this presentation, I will discuss recent studies of DNA polymerase holoenzyme assembly and exonuclease proofreading activity at p/t junctions. Specifically, results from microsecond-resolved polarization-sweep single-molecule spectroscopy (PS-SMS) demonstrate how protein-dependent conformational trapping of otherwise sparsely populated transient DNA local conformations is involved in processive holoenzyme formation.

Histone FRET microscopy of live cell genome architecture.

Inside the nucleus of an intact cell, DNA is folded around histone proteins into nucleosomes and compacted into a multi-layered three-dimensional chromatin network. The nanometre spacing between nucleosomes positioned throughout this structural framework is known to locally modulate local DNA template access and regulate genome function. However, given that this structural feature occurs on a spatial scale well below the diffraction limit of optical microscopy, real time observation of nucleosome proximity in live cells has proven technically difficult, despite recent advances in live cell super resolution imaging. Thus, in recent work, we have been exploring a powerful alternative solution that is based on fluorescence lifetime and anisotropy imaging microscopy (FLIM and FAIM) of Förster resonance energy transfer (FRET) between fluorescently labelled histones - the core protein of a nucleosome. Here we present recent findings from application of this technology to the study of live cell chromatin network dynamics, which demonstrate local ensemble nucleosome proximity, spatiotemporally oscillates between different spacings that are ‘invisible’ to diffraction limited microscopy.

Studies of Local DNA Backbone Conformation and Conformational Disorder Using Site-Specific Exciton-Coupled Dimer Probe Spectroscopy.

The processes of genome expression, regulation, and repair require direct interactions between proteins and DNA at specific sites located at and near single-stranded-double-stranded DNA (ssDNA-dsDNA) junctions. Here, we review the application of recently developed spectroscopic methods and analyses that combine linear absorbance and circular dichroism spectroscopy with nonlinear 2D fluorescence spectroscopy to study the local conformations and conformational disorder of the sugar-phosphate backbones of ssDNA-dsDNA fork constructs that have been internally labeled with exciton-coupled cyanine (iCy3)2 dimer probes. With the application of these methods, the (iCy3)2 dimer can serve as a reliable probe of the mean local conformations and conformational distributions of the sugar-phosphate backbones of dsDNA at various critical positions. The results of our studies suggest a possible structural framework for understanding the roles of DNA breathing in driving the processes of protein-DNA complex assembly and function.

Sequence-specific binding behavior of coralyne toward triplex DNA: An ultrafast time-resolved fluorescence spectroscopy study.

Triplex DNA structure has potential therapeutic application in inhibiting the expression of genes involved in cancer and other diseases. As a DNA-targeting antitumor and antibiotic drug, coralyne shows a remarkable binding propensity to triplex over canonical duplex and thus can modulate the stability of triplex structure, providing a prospective gene targeting strategy. Much less is known, however, about coralyne-binding interactions with triplex. By combining multiple steady-state spectroscopy with ultrafast fluorescence spectroscopy, we have investigated the binding behaviors of coralyne with typical triplexes. Upon binding with a G-containing triplex, the fluorescence of coralyne is markedly quenched owing to the photoinduced electron transfer (PET) of coralyne with the G base. Systematic studies show that the PET rates are sensitive to the binding configuration and local microenvironment, from which the coexisting binding modes of monomeric (full and partial) intercalation and aggregate stacking along the sugar-phosphate backbone are distinguished and their respective contributions are determined. It shows that coralyne has preferences for monomeric intercalation within CGG triplex and pure TAT triplex, whereas CGC+ triplex adopts mainly backbone binding of coralyne aggregates due to charge repulsion, revealing the sequence-specific binding selectivity. The triplex-DNA-induced aggregation of coralyne could be used as a probe for recognizing the water content in local DNA structures. The strong π-π stacking of intercalated coralyne monomer with base-triplets plays an important role in stabilizing the triplex structure. These results provide mechanistic insights for understanding the remarkable propensity of coralyne in selective binding to triplex DNA and shed light on the prospective applications of coralyne-triplex targeted anti-gene therapeutics.