Stretches Of DNA Research Articles

Action-At-A-Distance in DNA Mismatch Repair: Mechanistic Insights and Models for How DNA and Repair Proteins Facilitate Long-Range Communication

Many DNA metabolic pathways, including DNA repair, require the transmission of signals across long stretches of DNA or between DNA molecules. Solutions to this signaling challenge involve various mechanisms: protein factors can travel between these sites, loop DNA between sites, or form oligomers that bridge the spatial gaps. This review provides an overview of how these paradigms have been used to explain DNA mismatch repair, which involves several steps that require action-at-a-distance. Here, we describe these models in detail and how current data fit into these descriptions. We also outline regulation steps that remain unanswered in how the action is communicated across long distances along a DNA contour in DNA mismatch repair.

53BP1-the ‘Pandora’s box’ of genome integrity

53BP1 has several functions in the maintenance of genome integrity. It functions as a key mediator involved in double-strand break (DSB) repair, which functions to maintain a balance in the repair pathway choices and in preserving genomic stability. While its DSB repair functions are relatively well-characterized, its role in DNA replication and replication fork protection is less understood. In response to replication stress, 53BP1 contributes to fork protection by regulating fork reversal and restart. It helps maintain replication fork stability and speed, with 53BP1 loss leading to defective fork progression and increased sensitivity to replication stress agents. However, 53BP1's precise role in fork protection remains debated, as some studies have not observed protective effects. Therefore, it is critical to determine the role of 53BP1 in replication to better understand when it promotes replication fork protection, and the underlying mechanisms involved. Moreover, 53BP1's function in replication stress extends beyond its activity at active replication forks; it also forms specialized nuclear bodies (NBs) which protect stretches of under-replicated DNA (UR-DNA) transmitted from a previous cell cycle to daughter cells through mitosis. The mechanism of 53BP1 NBs in the coordination of replication and repair events at UR-DNA loci is not fully understood and warrants further investigation. The present review article focuses on elucidating 53BP1’s functions in replication stress (RS), its role in replication fork protection, and the significance of 53BP1 NBs in this context to provide a more comprehensive understanding of its less well-established role in DNA replication.

Plasmids affect microindel mutations in Acinetobacter baylyi ADP1

Plasmids can impact the evolution of their hosts, e.g. due to carriage of mutagenic genes, through cross-talk with host genes or as result of SOS induction during transfer. Here we demonstrate that plasmids can affect the level of microindel mutations in the host genome. These mutations are driven by the production of single-stranded DNA molecules that invade replication forks at microhomologies and subsequently get integrated into the genome. Using the gammaproteobacterial model organism Acinetobacter baylyi, we show that carriage of broad host range plasmids from different incompatibility groups can cause microindel mutations directly or indirectly. The plasmid vector pQLICE belonging to the incompatibility group Q (IncQ) and replicating by a characteristic strand displacement mechanism can generate chromosomal microindel mutations directly with short stretches of DNA originating from pQLICE. In addition, results with the IncP plasmid vector pRK415 (theta replication mechanism) show that the presence of plasmids can increase microindel mutation frequencies indirectly (i.e., with chromosomal ectopic DNA), presumably through plasmid-chromosome interactions that lead to DNA damages. These results provide new mechanistic insights into the microindel mutation mechanism, suggesting that single-stranded DNA repair intermediates are the causing agents. By contrast, the IncN plasmid RN3 appears to suppress host microindel mutations. The suppression mechanism remains unknown. Other plasmids in this study (belonging to IncA/C2, IncW, pBBR incompatibility groups) confer ambiguous or no quantifiable mutagenic effects.

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is theCDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Prediction and functional interpretation of inter-chromosomal genome architecture from DNA sequence with TwinC.

Three-dimensional nuclear DNA architecture comprises well-studied intra-chromosomal (cis) folding and less characterized inter-chromosomal (trans) interfaces. Current predictive models of 3D genome folding can effectively infer pairwise cis-chromatin interactions from the primary DNA sequence but generally ignore trans contacts. There is an unmet need for robust models of trans-genome organization that provide insights into their underlying principles and functional relevance. We present TwinC, an interpretable convolutional neural network model that reliably predicts trans contacts measurable through genome-wide chromatin conformation capture (Hi-C). TwinC uses a paired sequence design from replicate Hi-C experiments to learn single base pair relevance in trans interactions across two stretches of DNA. The method achieves high predictive accuracy (AUROC=0.80) on a cross-chromosomal test set from Hi-C experiments in heart tissue. Mechanistically, the neural network learns the importance of compartments, chromatin accessibility, clustered transcription factor binding and G-quadruplexes in forming trans contacts. In summary, TwinC models and interprets trans genome architecture, shedding light on this poorly understood aspect of gene regulation.

Effects of PCNA Stability on the Formation of Mutations.

The fidelity of replication, especially in the presence of DNA damage, is essential for the proper function of cells. Mutations that inactivate genes involved in DNA damage repair or bypass are enriched in several types of cancer cells. Thus, it is important to further our understanding of the mechanisms governing replication fidelity. PCNA is a ring-shaped complex that encircles DNA at the front of the replication fork, at the double-stranded/single-stranded DNA junction. It serves as a processivity factor for the different DNA replication polymerases, allowing them to replicate longer stretches of DNA by physically tethering them to the DNA and preventing their detachment. In addition, PCNA also regulates and coordinates different DNA damage bypass pathways meant to allow DNA replication in the presence of DNA damage. Due to its essentiality and the numerous functions it has in the cell, much is still unclear about PCNA. Here, we utilize PCNA mutants that lower the stability of the PCNA complex on the chromatin, and thus tend to disassociate and fall from the DNA. Using these mutants, we show that PCNA's physical presence on the DNA can prevent DNA misalignment at repetitive sequences, leading to increased mutation formation. We also show that PCNA-interacting proteins play an important role in strengthening the ring's stability on the chromatin. Such repetitive sequence-induced mutations are common in several human diseases and it is important to study their formation and the mechanisms guarding against them.

Structural determinants of DNA cleavage by a CRISPR HNH-Cascade system

Canonical prokaryotic type I CRISPR-Cas adaptive immune systems contain a multicomponent effector complex called Cascade, which degrades large stretches of DNA via Cas3 helicase-nuclease activity. Recently, a highly precise subtype I-F1 CRISPR-Cas system (HNH-Cascade) was found that lacks Cas3, the absence of which is compensated for by the insertion of an HNH endonuclease domain in the Cas8 Cascade component. Here, we describe the cryo-EM structure of Selenomonas sp. HNH-Cascade (SsCascade) in complex with target DNA and characterize its mechanism of action. The Cascade scaffold is complemented by the HNH domain, creating a ring-like structure in which the unwound target DNA is precisely cleaved. This structure visualizes a unique hybrid of two extensible biological systems-Cascade, an evolutionary platform for programmable DNA effectors, and an HNH nuclease, an adaptive domain with a spectrum of enzymatic activity.

The translesion polymerase Pol Y1 is a constitutive component of the B. subtilis replication machinery.

Unrepaired DNA damage encountered by the cellular replication machinery can stall DNA replication, ultimately leading to cell death. In the DNA damage tolerance pathway translesion synthesis (TLS), replication stalling is alleviated by the recruitment of specialized polymerases to synthesize short stretches of DNA near a lesion. Although TLS promotes cell survival, most TLS polymerases are low-fidelity and must be tightly regulated to avoid harmful mutagenesis. The gram-negative bacterium Escherichia coli has served as the model organism for studies of the molecular mechanisms of bacterial TLS. However, it is poorly understood whether these same mechanisms apply to other bacteria. Here, we use in vivo single-molecule fluorescence microscopy to investigate the TLS polymerase Pol Y1 in the model gram-positive bacterium Bacillus subtilis. We find significant differences in the localization and dynamics of Pol Y1 in comparison to its E. coli homolog, Pol IV. Notably, Pol Y1 is constitutively enriched at or near sites of replication in the absence of DNA damage through interactions with the DnaN clamp; in contrast, Pol IV has been shown to be selectively enriched only upon replication stalling. These results suggest key differences in the roles and mechanisms of regulation of TLS polymerases across different bacterial species.

Generating information-dense promoter sequences with optimal string packing.

Dense arrangements of binding sites within nucleotide sequences can collectively influence downstream transcription rates or initiate biomolecular interactions. For example, natural promoter regions can harbor many overlapping transcription factor binding sites that influence the rate of transcription initiation. Despite the prevalence of overlapping binding sites in nature, rapid design of nucleotide sequences with many overlapping sites remains a challenge. Here, we show that this is an NP-hard problem, coined here as the nucleotide String Packing Problem (SPP). We then introduce a computational technique that efficiently assembles sets of DNA-protein binding sites into dense, contiguous stretches of double-stranded DNA. For the efficient design of nucleotide sequences spanning hundreds of base pairs, we reduce the SPP to an Orienteering Problem with integer distances, and then leverage modern integer linear programming solvers. Our method optimally packs sets of 20-100 binding sites into dense nucleotide arrays of 50-300 base pairs in 0.05-10 seconds. Unlike approximation algorithms or meta-heuristics, our approach finds provably optimal solutions. We demonstrate how our method can generate large sets of diverse sequences suitable for library generation, where the frequency of binding site usage across the returned sequences can be controlled by modulating the objective function. As an example, we then show how adding additional constraints, like the inclusion of sequence elements with fixed positions, allows for the design of bacterial promoters. The nucleotide string packing approach we present can accelerate the design of sequences with complex DNA-protein interactions. When used in combination with synthesis and high-throughput screening, this design strategy could help interrogate how complex binding site arrangements impact either gene expression or biomolecular mechanisms in varied cellular contexts.

Oxidative Defect Detection Within Free and Packed DNA Systems: A Quantum Mechanical/Molecular Mechanics (QM/ MM) Approach.

Base excision repair enzymes (BERs) detect and repair oxidative DNA damage with efficacy despite the small size of the defects and their often only minor structural impact. A charge transfer (CT) model for rapid scanning of DNA stretches has been evoked to explain the high detection rate in the face of numerous, small lesions. The viability of CT DNA defect detection is explored via hybrid QM/MM computational studies that leverage the accuracy of quantum mechanics (QM) for a region of interest and the descriptive power of molecularmechanics (MM) for the remainder of the system. We find that the presence of an oxidative lesion lowers theredox free energy of oxidation by approximately 1.0 eV regardless of DNA compaction (free DNA versus packed DNA in nucleosome core particles) and damage location indicating the high feasibility of a CT-based process for defect detection in DNA.

Examining chromatin heterogeneity through PacBio long-read sequencing of M.EcoGII methylated genomes: an m6A detection efficiency and calling bias correcting pipeline.

Recent studies have combined DNA methyltransferase footprinting of genomic DNA in nuclei with long-read sequencing, resulting in detailed chromatin maps for multi-kilobase stretches of genomic DNA from one cell. Theoretically, nucleosome footprints and nucleosome-depleted regions can be identified using M.EcoGII, which methylates adenines in any sequence context, providing a high-resolution map of accessible regions in each DNA molecule. Here, we report PacBio long-read sequence data for budding yeast nuclei treated with M.EcoGII and a bioinformatic pipeline which corrects for three key challenges undermining this promising method. First, detection of m6A in individual DNA molecules by the PacBio software is inefficient, resulting in false footprints predicted by random gaps of seemingly unmethylated adenines. Second, there is a strong bias against m6A base calling as AT content increases. Third, occasional methylation occurs within nucleosomes, breaking up their footprints. After correcting for these issues, our pipeline calculates a correlation coefficient-based score indicating the extent of chromatin heterogeneity within the cell population for every gene. Although the population average is consistent with that derived using other techniques, we observe a wide range of heterogeneity in nucleosome positions at the single-molecule level, probably reflecting cellular chromatin dynamics.

Amplification of different satellite-DNAs in prostate cancer

In various solid tumors and corresponding cell lines, prior research has identified acquired copy number variations (CNVs) encompassing centromeric satellite-DNA sequences. This observation emerged from the application of centromeric probes (satellite-DNA) as controls in molecular cytogenetic investigations and diagnostics, although these accounts were largely anecdotal. In this study, we conducted a systematic screening for satellite-DNA sequence amplification in 31 prostate cancer (PCa) samples, a prevalent malignancy in men characterized by discernible molecular cytogenetic aberrations. Notably, PCa-typical genetic aberrations, such as TMPRSS2-ERG gene rearrangements and PTEN deletion, were identified in 12 and 6 out of the 31 PCa samples, respectively. Overall, PCa exhibited genomic instability marked by chromosomal gain or loss of signals across nearly all tested satellite-DNA regions, with particular emphasis on the Y-chromosome (18/31 cases). Remarkably, 5/12 PCa samples representing more advanced metastatic cancer displayed amplification of one or two satellite DNA stretches each, being detectable as blocks analogous to homogenously staining regions. Notably, these stretches included α-satellite DNA derived from chromosomes 2, 3, 4, 15, and 20, as well as satellite-III DNAs (D1Z1 and DYZ1). These findings align with recent discoveries indicating that α-satellite DNAs are expressed as long-non-coding RNAs in advanced cancer, particularly in the context of PCa.

The genome organization of the Lake Magadi tilapia, Oreochromis Alcolapia grahami, a cichlid extremophile

The genome of vertebrates is made of a mosaic of long stretches of DNA, called isochores, which are compositionally uniform, and belong to a few families of GC-poor (L1 and L2) and GC-rich (H1, H2, and H3) components. Poikilotherms tend to have GC-poor genomes, while endotherms comprise both GC-poor and GC-rich isochores. The thermal theory claimed that temperature and natural selection played an active role in favoring GC-rich genomic regions, yet empirical evidence was difficult to obtain. Early work based on cesium chloride ultracentrifugation gradients showed that the Lake Magadi tilapia, a hot-water adapted fish species, displayed GC-rich regions that were absent from a close relative that lives in colder water. The goal of this study was to revisit the original study using full genome sequencing. We found that the original GC-rich regions are indeed present, that they are interspersed in the genome. Indeed, when comparing Lake Magadi tilapia with the temperate Nile tilapia, we found that 59.3 % of the genome of Lake Magadi tilapia had a base composition higher than 40 %GC, as opposed to 55.3 % of the genome of the Nile tilapia having a base composition higher than 40 % GC. We also found that their genomes comprised similar amounts of repetitive elements (20 % and 19.5 %, respectively) indicating that the shifts in base composition might not be due to repetitive elements. Further work on repetitive element analyses, protein coding genes and additional hot-water adapted fishes will provide clues as to the origin of GC-rich isochores in Lake Magadi tilapia.

Performance of a 74-Microhaplotype Assay in Kinship Analyses.

Microhaplotypes (MHs) consisting of multiple SNPs and indels on short stretches of DNA are new and interesting loci for forensic genetic investigations. In this study, we analysed 74 previously defined MHs in two of the populations that our laboratory provides with forensic genetic services, Danes and Greenlanders. In addition to the 229 SNPs that originally made up the 74 MHs, 66 SNPs and 3 indels were identified in the two populations, and 45 of these variants were included in new definitions of the MHs, whereas 24 SNPs were considered rare and of little value for case work. The average effective number of alleles (Ae) was 3.2, 3.0, and 2.6 in Danes, West Greenlanders, and East Greenlanders, respectively. High levels of linkage disequilibrium were observed in East Greenlanders, which reflects the characteristics of this population that has a small size, and signs of admixture and substructure. Pairwise kinship simulations of full siblings, half-siblings, first cousins, and unrelated individuals were performed using allele frequencies from MHs, STRs and SNPs from Danish and Greenlandic populations. The MH panel outperformed the currently used STR and SNP marker sets and was able to differentiate siblings from unrelated individuals with a 0% false positive rate and a 1.1% false negative rate using an LR threshold of 10,000 in the Danish population. However, the panel was not able to differentiate half-siblings or first cousins from unrelated individuals. The results generated in this study will be used to implement MHs as investigative markers for relationship testing in our laboratory.

Comparative Analysis of Predicted SSR Sequences and CpG Islands to Discover Evolutionary Relics of Sex-chromosomes in Divergent Animal Species

Background: DNA markers have high occurrence and mutation rates and are generally located around the controlling regions of some tissue-specific genes and housekeeping genes that can change the expression pattern. Microsatellites and CpG islands are stretches of DNA with repeats and are known to influence gene expression. Methods: In the present study, these DNA markers are mined and an In silico comparison was carried out to understand their occurrence pattern and distribution frequency in sex chromosomes (X and Y) of 12 different animal species using Perl and R programming pipelines. Result: It was found that female-dominant X chromosomes had higher occurrence and distribution frequencies for these DNA markers than that of male-dominant sex chromosome i.e. Y which means that the former has a higher number of the evolutionary sites.The density of DNA markers however, showed remarkable variation for different animal species. The results obtained need validation through wet-lab experimentation. Tri- and hexa-nucleotide repeats are more abundant in exons, whereas other repeats are more abundant in non-coding regions.

Mechanism analysis of toxic and side effects of chemicals on human body

The exposure of the human body to different chemical substances poses a critical hazard to public well-being. Chemical toxicity is fundamentally ascribed to the disturbance of typical biological processes and can result in unfavorable health results. Understanding the components by which chemical substances apply their harmful impacts is vital for surveying and moderating these dangers. In this research, it will discuss the different routes of exposure, such as inhalation ingestion and dermal contact through which chemicals can enter the body. This research investigates the complex instruments at the atomic and cellular levels, counting oxidative stretch DNA harm and disturbance of enzymatic pathways by which harmful impacts are actuated. The evaluation of chemical toxicity includes a range of toxicological considerations, including acute and chronic toxicity tests, genotoxicity measures, and carcinogenicity evaluations. The development of reliable toxicity evaluation strategies, such as in vitro and in silico models, plays an urgent part in predicting the adverse impacts of chemical substances. Administrative systems and hazard evaluation techniques are discussed to guarantee peoples and the environments security. A comprehensive understanding of the components and assessment of poisonous side impacts is essential for defending open health and minimizing the hindering effects of chemical exposure.

Spatial and temporal characterization of the rich fraction of plastid DNA present in the nuclear genome of Moringa oleifera reveals unanticipated complexity in NUPTs´ formation

BackgroundBeyond the massive amounts of DNA and genes transferred from the protoorganelle genome to the nucleus during the endosymbiotic event that gave rise to the plastids, stretches of plastid DNA of varying size are still being copied and relocated to the nuclear genome in a process that is ongoing and does not result in the concomitant shrinking of the plastid genome. As a result, plant nuclear genomes feature small, but variable, fraction of their genomes of plastid origin, the so-called nuclear plastid DNA sequences (NUPTs). However, the mechanisms underlying the origin and fixation of NUPTs are not yet fully elucidated and research on the topic has been mostly focused on a limited number of species and of plastid DNA.ResultsHere, we leveraged a chromosome-scale version of the genome of the orphan crop Moringa oleifera, which features the largest fraction of plastid DNA in any plant nuclear genome known so far, to gain insights into the mechanisms of origin of NUPTs. For this purpose, we examined the chromosomal distribution and arrangement of NUPTs, we explicitly modeled and tested the correlation between their age and size distribution, we characterized their sites of origin at the chloroplast genome and their sites of insertion at the nuclear one, as well as we investigated their arrangement in clusters. We found a bimodal distribution of NUPT relative ages, which implies NUPTs in moringa were formed through two separate events. Furthermore, NUPTs from every event showed markedly distinctive features, suggesting they originated through distinct mechanisms.ConclusionsOur results reveal an unanticipated complexity of the mechanisms at the origin of NUPTs and of the evolutionary forces behind their fixation and highlight moringa species as an exceptional model to assess the impact of plastid DNA in the evolution of the architecture and function of plant nuclear genomes.

Progress in polymerase chain reaction technology and its contribution to the tuberculosis diagnostic ecosystem in India: A mini review

The advent of Polymerase Chain Reaction (PCR) has revolutionized the human molecular diagnostic scenario. Its ability to exponentially generate desired stretch of DNA from chromosomal or complimentary DNA has laid the foundation for sensitive and specific detection of a pathogen or an aberrant stretch of DNA. Importantly, PCR technology has helped generate diagnostic immunity from phenotypic variation of pathogenic targets. The technological evolution of conventional end-point PCR to its real time version further added clinically important features such as enhanced sensitivity and quantification of target DNA molecules. Two important chemistries became prominent in the real-time PCR domain. The intercalating dye-based chemistry comes with convenience, economy and feature such as melt curve analysis but also carries the disadvantage of overestimation of signal due to unwanted signal from primer dimers. Dual labelled probe on the other hand provides unprecedented sensitivity and specificity. Isothermal method of genome nucleic acid amplification has also evolved as a parallel and convenient method of amplification-based detection of nucleic acid targets. Tuberculosis diagnostics has immensely benefitted from these technologies. However, unmet need exists in sputum handling methods and in its adaptation for molecular diagnostics in resource limited settings. Significant dependence for diagnostic needs on the western world is a matter of concern in India. The country will benefit from rapid and indigenous research and development of molecular diagnostic solutions by way of formulating kits for rapid extraction of DNA and RNA from various clinical sources and developing detection technologies that can compete international products in its category. A solution to address sputum transportation, storage and DNA extraction that has enhanced advantages compared to current methods will also be helpful in the efforts to make India free from tuberculosis.

Statistical analysis of synonymous and stop codons in pseudo-random and real sequences as a function of GC content

Knowledge of the frequencies of synonymous triplets in protein-coding and non-coding DNA stretches can be used in gene finding. These frequencies depend on the GC content of the genome or parts of it. An example of interest is provided by stop codons. This is relevant for the definition of Open Reading Frames. A generic case is provided by pseudo-random sequences, especially when they code for complex proteins or when they are non-coding and not subject to selection pressure. Here, we calculate, for such sequences and for all 25 known genetic codes, the frequency of each amino acid and stop codon based on their set of codons and as a function of GC content. The amino acids can be classified into five groups according to the GC content where their expected frequency reaches its maximum. We determine the overall Shannon information based on groups of synonymous codons and show that it becomes maximum at a percent GC of 43.3% (for the standard code). This is in line with the observation that in most fungi, plants, and animals, this genomic parameter is in the range from 35 to 50%. By analysing natural sequences, we show that there is a clear bias for triplets corresponding to stop codons near the 5′- and 3′-splice sites in the introns of various clades.

Yeast zinc cluster transcription factors involved in the switch from fermentation to respiration show interdependency for DNA binding revealing a novel type of DNA recognition.

In budding yeast, fermentation is the most important pathway for energy production. Under low-glucose conditions, ethanol is used for synthesis of this sugar requiring a shift to respiration. This process is controlled by the transcriptional regulators Cat8, Sip4, Rds2 and Ert1. We characterized Gsm1 (glucose starvation modulator 1), a paralog of Rds2 and Ert1. Genome-wide analysis showed that Gsm1 has a DNA binding profile highly similar to Rds2. Binding of Gsm1 and Rds2 is interdependent at the gluconeogenic gene FBP1. However, Rds2 is required for Gsm1 to bind at other promoters but not the reverse. Gsm1 and Rds2 also bind to DNA independently of each other. Western blot analysis revealed that Rds2 controls expression of Gsm1. In addition, we showed that the DNA binding domains of Gsm1 and Rds2 bind cooperatively in vitro to the FBP1 promoter. In contrast, at the HAP4 gene, Ert1 cooperates with Rds2 for DNA binding. Mutational analysis suggests that Gsm1/Rds2 and Ert1/Rds2 bind to short common DNA stretches, revealing a novel mode of binding for this class of factors. Two-point mutations in a HAP4 site convert it to a Gsm1 binding site. Thus, Rds2 controls binding of Gsm1 at many promoters by two different mechanisms: regulation of Gsm1 levels and increased DNA binding by formation of heterodimers.