DNA Translocation Research Articles

Surface exclusion of IncC conjugative plasmids and their relatives.

The phenomenon of exclusion allows conjugative plasmids to selectively impede the entry of identical or related elements into their host cell to prevent the resulting instability. Entry exclusion blocks DNA translocation into the recipient cell, whereas surface exclusion destabilizes the mating pair. IncC conjugative plasmids largely contribute to the dissemination of antibiotic-resistance genes in Gammaproteobacteria. IncC plasmids are known to exert exclusion against their relatives, including IncC and IncA plasmids, yet the entry exclusion factor eexC alone does not account for the totality of the exclusion phenotype. In this study, a transposon-directed insertion sequencing approach identified sfx as necessary and sufficient for the remaining exclusion phenotype. Sfx is an exclusion factor unrelated to the ones described to date. A cell fractionation assay localized Sfx in the outer membrane. Reverse transcription PCR and beta-galactosidase experiments showed that sfx is expressed constitutively at a higher level than eexC. A search in Gammaproteobacteria genomes identified Sfx homologs encoded by IncC, IncA and related, untyped conjugative plasmids and an uncharacterized family of integrative and mobilizable elements that likely rely on IncC plasmids for their mobility. Mating assays demonstrated that sfx is not required in the donor for exclusion, ruling out Sfx as the exclusion target. Instead, complementation assays revealed that the putative adhesin TraN in the donor mediates the specificity of surface exclusion. Mating assays with TraN homologs from related untyped plasmids from Aeromonas spp. and Photobacterium damselae identified two surface exclusion groups, with each Sfx being specific of TraN homologs from the same group. Together, these results allow us to better understand the apparent incompatibility between IncA and IncC plasmids and to propose a mechanistic model for surface exclusion mediated by Sfx in IncC plasmids and related elements, with implications for the rampant dissemination of antibiotic resistance.

Enhancing DNA Translocation Performance and Conformational Changes in Quartz Nanopipettes by Ionic Liquid Modification

Enhancing DNA Translocation Performance and Conformational Changes in Quartz Nanopipettes by Ionic Liquid Modification

Translocation of Gaussian polymers across a nanometric cylindrical channel

We present an analytical model to study translocation of Gaussian polymers across a cylindrical channel of nanometric size with a chemical potential inside the channel. Results show that polymer conformational entropy generates an entropic M-like free energy barrier for translocation. The presence of a small negative chemical potential reduces the entropic free energy barrier rendering the translocation time to follow a power law τ = AN ν as function of polymer size N. Power law exponents ν found here in varying the channel radius R, run from 1.525 to 1.999 for unforced translocation, and from 1.594 to 2.006 for translocation with small chemical potentials when R = 1 nm. Presence of large negative chemical potentials generate a free energy well rendering the translocation time to follow an exponential growth τ = Ae α N . We show existence of a negative chemical potential μ c that minimizes the translocation time due to an interplay of conformational entropy and channel-polymer interactions. The translocation time as function of channel length L grows exponentially as τ = Ae cL , it runs from milliseconds up to decades in the range of lengths found in biological channels. Interestingly, small negative chemical potentials approaching μ c overcome the effect of large channel lengths reducing the translocation time below seconds. Translocation speeds <v(N) > show a maximum of micrometers per second then it decays with polymer size and channel length, the characteristic decay <v(N) > ∼ N −1 has been observed in previous experiments of voltage-driven DNA translocation.

Differential contributions of double-strand break repair pathways to DNA rearrangements following the irradiation of Arabidopsis seeds and seedlings with ion beams.

DNA rearrangements, including inversions, translocations, and large insertions/deletions (indels), are crucial for crop evolution, domestication, and improvement. The rearrangements are frequently induced by ion beams via the mis-repair of DNA double-strand breaks (DSBs). Unfortunately, how ion beam-induced DSBs are repaired has not been comprehensively analyzed and the mechanisms underlying DNA rearrangements remain unclear. In this study, clonal sectors originating from single mutated cells in carbon ion-irradiated plants were used for whole-genome sequencing analyses after Arabidopsis seeds and seedlings were irradiated. Comparative analyses of the induced mutations (e.g., size and frequency of indels and microhomology at the junctions of the rearrangements) in the irradiated materials suggested that the broken/rejoined DSB ends were more extensively processed in seedlings than in seeds. A mutation to canonical non-homologous end-joining (c-NHEJ), which is a DSB repair pathway with minimal processing of DSB ends, increased the sensitivity to ion beams more in the seeds than in the seedlings, which was consistent with the junction analysis results, indicative of the minor contribution of c-NHEJ to the carbon ion-induced DSB repair in seedlings. Considering the characteristics of the large templated insertions in irradiated seedlings, ion-beam-induced DSBs in seedlings are likely repaired primarily by a polymerase theta-mediated pathway. Polymerase theta-deficient seedlings were more sensitive to ion beams than the c-NHEJ-deficient seedlings, consistent with this hypothesis. This study revealed the key characteristics of ion beam-induced DSBs and the associated repair mechanisms related to the physiological status of the irradiated materials, with implications for elucidating the occurrence and induction of rearrangements.

Strand dependent bypass of DNA lesions during fork reversal by ATP-dependent translocases SMARCAL1, ZRANB3, and HLTF.

During DNA replication, the replisome encounters obstacles including DNA lesions, transcription-replication conflicts, and other sources of replication stress. These obstacles must be efficiently overcome to complete DNA synthesis and minimize genome instability. One pathway to tolerate replication stress is replication fork reversal, in which parental template DNA strands are reannealed and a nascent-nascent DNA duplex is formed. Several enzymes promote replication fork reversal, including the ATP-dependent translocases SMARCAL1, ZRANB3, and HLTF. How these enzymes translocate on DNA that contains fork-stalling lesions is unknown. Here, we examined the abilities of SMARCAL1, ZRANB3, and HLTF to tolerate various lesions on leading or lagging template strands. We demonstrate that SMARCAL1 and ZRANB3 are selectively inhibited by lesions on the leading template strand, whereas HLTF is insensitive to bulky lesions on either strand. These results suggest that SMARCAL1 and ZRANB3 contact the leading strand during fork reversal and therefore are more sensitive to inhibition by bulky lesions on this strand. In contrast, HLTF DNA translocation is inherently insensitive to DNA lesions. These biochemical differences between the fork reversal enzymes provide insights into their mechanism of DNA remodeling and suggest they may act in lesion-specific contexts.

Helicase HELQ: Molecular Characters Fit for DSB Repair Function.

The protein sequence and spatial structure of DNA helicase HELQ are highly conserved, spanning from archaea to humans. Aside from its helicase activity, which is based on DNA binding and translocation, it has also been recently reconfirmed that human HELQ possesses DNA-strand-annealing activity, similar to that of the archaeal HELQ homolog StoHjm. These biochemical functions play an important role in regulating various double-strand break (DSB) repair pathways, as well as multiple steps in different DSB repair processes. HELQ primarily facilitates repair in end-resection-dependent DSB repair pathways, such as homologous recombination (HR), single-strand annealing (SSA), microhomology-mediated end joining (MMEJ), as well as the sub-pathways' synthesis-dependent strand annealing (SDSA) and break-induced replication (BIR) within HR. The biochemical functions of HELQ are significant in end resection and its downstream pathways, such as strand invasion, DNA synthesis, and gene conversion. Different biochemical activities are required to support DSB repair at various stages. This review focuses on the functional studies of the biochemical roles of HELQ during different stages of diverse DSB repair pathways.

Bacterial chromatin proteins, transcription, and DNA topology: Inseparable partners in the control of gene expression.

DNA in bacterial chromosomes is organized into higher-order structures by DNA-binding proteins called nucleoid-associated proteins (NAPs) or bacterial chromatin proteins (BCPs). BCPs often bind to or near DNA loci transcribed by RNA polymerase (RNAP) and can either increase or decrease gene expression. To understand the mechanisms by which BCPs alter transcription, one must consider both steric effects and the topological forces that arise when DNA deviates from its fully relaxed double-helical structure. Transcribing RNAP creates DNA negative (-) supercoils upstream and positive (+) supercoils downstream whenever RNAP and DNA are unable to rotate freely. This (-) and (+) supercoiling generates topological forces that resist forward translocation of DNA through RNAP unless the supercoiling is constrained by BCPs or relieved by topoisomerases. BCPs also may enhance topological stress and overall can either inhibit or aid transcription. Here, we review current understanding of how RNAP, BCPs, and DNA topology interplay to control gene expression.

Two residues in the DNA binding site of Pif1 helicase are essential for nuclear functions but dispensable for mitochondrial respiratory growth.

Pif1 helicase functions in both the nucleus and mitochondria. Pif1 tightly couples ATP hydrolysis, single-stranded DNA translocation, and duplex DNA unwinding. We investigated two Pif1 variants (F723A and T464A) that have each lost one site of interaction of the protein with the DNA substrate. Both variants exhibit minor reductions in affinity for DNA and ATP hydrolysis but have impaired DNA unwinding activity. However, these variants translocate on single-stranded DNA faster than the wildtype enzyme and can slide on the DNA substrate in an ATP-independent manner. This suggests they have lost their grip on the DNA, interfering with coupling ATP hydrolysis to translocation and unwinding. Yeast expressing these variants have increased gross chromosomal rearrangements, increased telomere length, and can overcome the lethality of dna2Δ, similar to phenotypes of yeast lacking Pif1. However, unlike pif1Δ mutants, they are viable on glycerol containing media and maintain similar mitochondrial DNA copy numbers as Pif1 wildtype. Overall, our data indicate that a tight grip of the trailing edge of the Pif1 enzyme on the DNA couples ATP hydrolysis to DNA translocation and DNA unwinding. This tight grip appears to be essential for the Pif1 nuclear functions tested but is dispensable for mitochondrial respiratory growth.

Signal Propagation in the ATPase Domain of Mycobacterium tuberculosis DNA Gyrase from Dynamical-Nonequilibrium Molecular Dynamics Simulations.

DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including Mycobacterium tuberculosis, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A2B2) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of M. tuberculosis GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of M. tuberculosis DNA gyrase relative to those from other bacterial species.

Selective Capture and Manipulation of DNA through Double Charged Nanopores.

In the past few decades, nanometer-scale pores have been employed as powerful tools for sensing biological molecules. Owing to its unique structure and properties, solid-state nanopores provide interesting opportunities for the development of DNA sequencing technology. Controlling DNA translocation in nanopores is an important means of improving the accuracy of sequencing. Here we present a proof of principle study of accelerating DNA captured across targeted graphene nanopores using surface charge density and find the intrinsic mechanism of the combination of electroosmotic flow induced by charges of nanopore and electrostatic attraction/repulsion between the nanopore and ssDNA. The theoretical study performed here provides a new means for controlling DNA transport dynamics and makes better and cheaper application of graphene in molecular sequencing.

Bacterial DNA Translocation Is Associated With Overt Hepatic Encephalopathy and Mortality in Patients With Cirrhosis.

Data on the relationship between bacterial translocation, hepatic encephalopathy (HE), and mortality are scarce. This study aimed to assess the association between bacterial DNA (bactDNA) translocation, inflammatory response, ammonia levels, and severity of HE in patients with cirrhosis, as well as the role of bactDNA translocation in predicting mortality. Cirrhotic patients without bacterial infection were prospectively enrolled between June 2022 and January 2023. Grading of HE was classified by the West Haven Criteria and Psychometric Hepatic Encephalopathy Score ≤ -5. Overall, 294 cirrhotic patients were enrolled, with 92 (31.3%) and 58 (19.7%) having covert and overt HE, respectively. BactDNA translocation was detected in 36.1% of patients (n = 106). Patients with overt HE had more bactDNA translocation and higher serum lipopolysaccharide-binding protein (LBP), tumor necrosis factor-α, interleukin-6 (IL-6), and ammonia levels than those without HE. Patients with detectable bactDNA had higher white cell counts and serum LBP and IL-6 levels than those without. By contrast, bactDNA, serum LBP, and soluble CD14 levels were comparable between patients with covert HE and those without HE. The multivariate Cox regression analysis revealed that bactDNA translocation (hazard ratio [HR] = 2.49, 95% confidence interval [CI]: 1.22-5.11), Model for End-Stage Liver Disease score (HR = 1.12, 95% CI: 1.09-1.16), age (HR = 1.05, 95% CI: 1.000-1.002), and baseline IL-6 (HR = 1.001, 95% CI: 1.000-1.002) were independent factors associated with 6-month mortality. Apart from hyperammonemia, bactDNA translocation is a possible factor associated with overt HE in cirrhotic patients. BactDNA translocation and IL-6 are independent factors associated with 6-month mortality.

Ion-Solvent Interactions under Confinement Hold the Key to Tuning the DNA Translocation Speeds in Polyelectrolyte-Functionalized Nanopores.

DNA sequencing and sensing using nanopore technology delves critically into the alterations in the measurable electrical signal as single-stranded DNA is drawn through a tiny passage. To make such precise measurements, however, slowing down the DNA in the tightly confined passage is a key requirement, which may be achieved by grafting the nanopore walls with a polyelectrolyte layer (PEL). This soft functional layer at the wall, under an off-design condition, however, may block the DNA passage completely, leading to the complete loss of output signal from the nanobio sensor. Whereas theoretical postulates have previously been put forward to explain the essential physics of DNA translocation in nanopores, these have turned out to be somewhat inadequate when confronted with the experimental findings on functionalized nanopores, including the prediction of the events of complete signal losses. Circumventing these constraints, herein we bring out a possible decisive role of the interplay between the inevitable variabilities in the ionic distribution along the nanopore axis due to its finite length as opposed to its idealized "infinite" limit as well as the differential permittivity of PEL and bulk solution that cannot be captured by the commonly used one-dimensional variant of the electrical double layer theory. Our analysis, for the first time, captures variations in the ionic concentration distribution across multidimensional physical space and delineates its impact on the DNA translocation characteristics that have hitherto remained unaddressed. Our results reveal possible complete blockages of DNA translocation as influenced by less-than-threshold permittivity values or greater-than-threshold grafting densities of the PEL. In addition, electrohydrodynamic blocking is witnessed due to the ion-selective nature of the nanopore at low ionic concentrations. Hence, our study establishes a functionally active regime over which the PEL layer in a finite-length nanopore facilitates controllable DNA translocation, enabling successful sequencing and sensing through the explicit modulation of translocation speed.

Bisphenols can promote antibiotic resistance by inducing metabolic adaptations and natural transformation

Whether bisphenols, as plasticizers, can influence bacterial uptake of antibiotic resistance genes (ARGs) in natural environment, as well as the underlying mechanism remains largely unknown. Our results showed that four commonly used bisphenols (bisphenol A, S, F, and AF) at their environmental relative concentrations can significantly promote transmission of ARGs by 2.97–3.56 times in Acinetobacter baylyi ADP1. Intriguingly, we observed ADP1 acquired resistance by integrating plasmids uptake and cellular metabolic adaptations other than through reactive oxygen species mediated pathway. Metabolic adaptations including upregulation of capsules polysaccharide biosynthesis and intracellularly metabolic enzymes, which enabled formation of thicker capsules for capturing free plasmids, and degradation of accumulated compounds. Simultaneously, genes encoding DNA uptake and translocation machinery were incorporated to enhance natural transformation of antibiotic resistance carrying plasmids. We further exposed aquatic fish to bisphenols for 120 days to monitor their long-term effects in aquatic environment, which showed that intestinal bacteria communities were dominated by a drug resistant microbiome. Our study provides new insight into the mechanism of enhanced natural transformation of ARGs by bisphenols, and highlights the investigations for unexpectedly-elevated antibiotic-resistant risks by structurally related environmental chemicals.

Abstract 2935: NGS panel customization for comprehensive genomic profiling

Abstract Introduction: We have developed a custom SureSelect Cancer design pipeline for the creation of target enrichment gene panels for comprehensive profiling of single nucleotide variations (SNP/INDEL), gene-level copy number variations (CNV), and DNA translocations (TL) as well as content for measuring of microsatellite instability (MSI) and tumor mutational burden (TMB). Methods: The performance of individual probes was measured for the 679-gene SureSelect Cancer CGP panel and the information used to generate a reference database. When creating a new design, the gene list is cross-referenced with the database and when applicable, the probe information is applied to the new design. If genes are included in the custom design that are not present in the database, a second design algorithm selects the optimal probe based on its sequence characteristics (% GC, homology, etc.). Content is selected based on the biomarker to be measured and can be defined based on gene name (SNV/indel and CNV) or genomic coordinates (SNV/indel, TL). Pre-defined content for MSI measurement can be added to any design to cover 288 microsatellite sites. The performance of these sites in determining MSI status was demonstrated by comparing to the MSI-PCR reference method. To assess the performance of the design methodology, designs of various sizes (50 to 650+ genes) and content type were generated and assayed on reference samples and FFPE with known biomarkers. Sequencing metrics were evaluated for capture specificity and coverage of the target regions. Results: The design methodology allowed for sub-selection of content present in the 679-gene SureSelect Cancer CGP assay as well as addition of de-novo content. The design algorithm uses empirical data on probe performance to provide robust and reproducible results for the range of supported design sizes. Biomarkers were reliably detected with SNV detection down to 1% allele frequency, detection of gene amplifications and translocations concordant with FISH, and MSI and TMB determination concordant with PCR and Exome sequencing respectively. The design workspace was implemented in the SureDesign platform, providing a web-based interface for creating the designs. Conclusions: We demonstrated full customization capability of the SureSelect Cancer target enrichment platform that pairs user-defined content with an optimized probe design methodology to enable the sensitive detection of the common types of DNA alterations found in FFPE samples. Citation Format: Michael Ruvolo, Akanksha Khare, Nishita Matcha-Balaguruvappa, Katherine Wilkins, Linus Forsmark, Swati Gupta. NGS panel customization for comprehensive genomic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2935.

From isotopically labeled DNA to fluorescently labeled dynamic pili: building a mechanistic model of DNA transport to the cytoplasmic membrane.

SUMMARYNatural competence, the physiological state wherein bacteria produce proteins that mediate extracellular DNA transport into the cytosol and the subsequent recombination of DNA into the genome, is conserved across the bacterial domain. DNA must successfully translocate across formidable permeability barriers during import, including the cell membrane(s) and the cell wall, that are normally impermeable to large DNA polymers. This review will examine the mechanisms underlying DNA transport from the extracellular space to the cytoplasmic membrane. First, the challenges inherent to DNA movement through the cell periphery will be discussed to provide context for DNA transport during natural competence. The following sections will trace the development of a comprehensive model for DNA translocation to the cytoplasmic membrane, highlighting the crucial studies performed over the last century that have contributed to building contemporary DNA import models. Finally, this review will conclude by reflecting on what is still unknown about the process and the possible solutions to overcome these limitations.

Pulley Effect in the Capture of DNA Translocation through Solid-State Nanopores.

Nanopores are powerful single-molecule sensors for analyzing biomolecules such as DNA and proteins. Understanding the dynamics of DNA capture and translocation through nanopores is essential for optimizing their performance. In this study, we examine the effects of applied voltage and pore diameter on current blockage, translocation time, collision, and capture location by translocating λ-DNA through 5.7 and 16 nm solid-state nanopores. Ionic current changes are used to infer DNA conformations during translocation. We find that translocation time increases with pore diameter, which can be attributed to the decrease of the stall force. Linear and exponential decreases of collision frequency with voltage are observed in the 16 and 5.7 nm pores, respectively, indicating a free energy barrier in the small pore. Moreover, the results reveal a voltage-dependent bias in the capture location toward the DNA ends, which is explained by a "pulley effect" deforming the DNA as it approaches the pore. This study provides insights into the physics governing DNA capture and translocation, which can be useful for promoting single-file translocation to enhance nanopore sensing.

Structural insights into histone exchange by human SRCAP complex

Histone variant H2A.Z is found at promoters and regulates transcription. The ATP-dependent chromatin remodeler SRCAP complex (SRCAP-C) promotes the replacement of canonical histone H2A–H2B dimer with H2A.Z–H2B dimer. Here, we determined structures of human SRCAP-C bound to H2A-containing nucleosome at near-atomic resolution. The SRCAP subunit integrates a 6-subunit actin-related protein (ARP) module and an ATPase-containing motor module. The ATPase-associated ARP module encircles half of the nucleosome along the DNA and may restrain net DNA translocation, a unique feature of SRCAP-C. The motor module adopts distinct nucleosome binding modes in the apo (nucleotide-free), ADP-bound, and ADP-BeFx-bound states, suggesting that ATPase-driven movement destabilizes H2A–H2B by unwrapping the entry DNA and pulls H2A–H2B out of nucleosome through the ZNHIT1 subunit. Structure-guided chromatin immunoprecipitation sequencing analysis confirmed the requirement of H2A-contacting ZNHIT1 in maintaining H2A.Z occupancy on the genome. Our study provides structural insights into the mechanism of H2A-H2A.Z exchange mediated by SRCAP-C.

Membrane fission during bacterial spore development requires cellular inflation driven by DNA translocation

Membrane fission during bacterial spore development requires cellular inflation driven by DNA translocation

E. coli RecBCD nuclease domain regulates helicase activity but not single-stranded DNA translocation activity

E. coli RecBCD nuclease domain regulates helicase activity but not single-stranded DNA translocation activity

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

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