Biology subjects

Ha, T.

Publications and source records attributed to Ha, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Reliable amplification of highly repetitive or low complexity sequence DNA enabled by superhelicase-mediated isothermal amplification

PCR is a cornerstone of molecular biology, but many biologically important DNA templates remain difficult to amplify. Long tandem repeats, low-complexity tracts, and sequences with extreme base composition often yield low product levels, smeared bands, stutter products, or truncated amplicons. These failures can arise because repeated cycles of thermal denaturation and reannealing promote off-register annealing, polymerase slippage, secondary-structure formation, and incomplete extension. Previously, we developed SSB-Helicase Assisted Rapid PCR (SHARP), an isothermal amplification method in which an engineered superhelicase and single-stranded DNA-binding protein replace the thermal melting step of PCR with enzymatic strand separation. Here, we tested whether SHARP can improve amplification of templates that are refractory to conventional PCR. SHARP robustly amplified up to six identical tandem repeats of the Widom 601 nucleosome-positioning sequence and up to 35 identical ankyrin repeats, targets that were poorly amplified by conventional PCR under the conditions tested. SHARP also amplified templates with extreme base composition, including a 95% AT-rich template and GC-rich templates (up to ~95% GC), as well as a 99-repeat CGG tract associated with Fragile X syndrome and a CAG/CAA repeat tract from mutant HTT exon 1 associated with Huntington disease. Together, these results show that helicase-driven isothermal amplification can expand access to repetitive and compositionally extreme DNA sequences that are challenging for conventional thermocycling-based PCR.

bioengineering↗

bioRxiv2024

Rad52 sorts and stacks Rad51 at the DNA junction to promote homologous recombination

Homologous recombination (HR) repairs double-stranded DNA breaks (DSBs). The DSBs are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Rad51 is a recombinase and catalyzes strand invasion and the search for homology. However, it binds to ssDNA with lower affinity than RPA. Thus, mediator proteins such as Rad52/BRCA2 are required to promote Rad51 binding to RPA-coated ssDNA, but the underlying mechanisms remain poorly understood. Saccharomyces cerevisiae Rad52 interacts with Rad51 through two distinct binding modes. We here uncover that the Rad51-binding site in the disordered C-terminus of Rad52 (mode-1) sorts polydisperse Rad51 into discrete monomers. The second Rad51 binding site resides in the ordered N-terminal ring of Rad52 (mode-2), but this interaction occurs at only one position on the ring. In single molecule confocal fluorescence microscopy combined with optical tweezer analysis, we directly visualize filament formation using fluorescent-Rad51. Rad52 catalyzes Rad51 loading onto RPA-coated ssDNA, with a distinct preference for junctions, but no filament growth is observed. Deletion of the C-terminus of Rad52 results in loss of Rad51 sorting and abrogates Rad51 binding to RPA-coated DNA. While BRCA2 and Rad52 are structurally unrelated, many of these functional features are conserved. We describe a concerted Sort & Stack mechanism for mediator proteins in promoting HR.

biochemistry↗

bioRxiv2024

A high throughput single molecule platform to study DNA supercoiling effect on protein-DNA interactions

DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced negative and positive supercoiling in these plasmids using gyrase and reverse gyrase, respectively. Comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed both negative and positive DNA supercoiling enhances the binding interaction between MutS and a mismatched base pair but does not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also opens new avenues for exploring the intricate dynamics of protein-DNA interactions under the influences of supercoiling.

biophysics↗

bioRxiv2024

MARK2/MARK3 kinases are catalytic co-dependencies of YAP/TAZ in human cancer

The Hippo signaling pathway is commonly dysregulated in human cancer, which leads to a powerful tumor dependency on the YAP/TAZ transcriptional coactivators. Here, we used paralog co-targeting CRISPR screens to identify the kinases MARK2/3 as absolute catalytic requirements for YAP/TAZ function in diverse carcinoma and sarcoma contexts. Underlying this observation is direct MARK2/3-dependent phosphorylation of NF2 and YAP/TAZ, which effectively reverses the tumor suppressive activity of the Hippo module kinases LATS1/2. To simulate targeting of MARK2/3, we adapted the CagA protein from H. pylori as a catalytic inhibitor of MARK2/3, which we show exerts anti-tumor activity in vivo. Together, these findings reveal MARK2/3 as powerful co-dependencies of YAP/TAZ in human cancer; targets that may allow for pharmacology that restores Hippo pathway-mediated tumor suppression.

cancer biology↗

bioRxiv2024

Host response during unresolved urinary tract infection alters mammary tissue homeostasis through collagen deposition and TIMP1

Exposure to pathogens throughout a lifetime influences immunity and organ function. Here, we explored how the systemic host-response to bacterial urinary tract infection (UTI) induces tissue-specific alterations to the mammary gland. Utilizing a combination of histological tissue analysis, single cell RNA sequencing and flow cytometry, we identified that mammary tissue from UTI-bearing mice display collagen deposition, enlarged ductal structures, ductal hyperplasia with atypical epithelial transcriptomes and altered immune composition. Bacterial cells were absent in the mammary tissue and blood of UTI-bearing mice, therefore, alterations to the distal mammary tissue were mediated by the systemic host response to local infection. Furthermore, broad spectrum antibiotic treatment resolved the infection and restored mammary cellular and tissue homeostasis. Systemically, unresolved UTI correlated with increased plasma levels of the metalloproteinase inhibitor, TIMP1, which controls extracellular matrix (ECM) remodeling and neutrophil function. Treatment of nulliparous and post-lactation UTI-bearing female mice with a TIMP1 neutralizing antibody, or broad-spectrum antibiotic, prevented mammary collagen deposition, thus providing evidence for an unexpected link between the systemic host response during UTI and mammary alterations. SummaryThe systemic response during urinary tract infection induces TIMP1-driven collagen deposition specifically into the mammary gland.

developmental biology↗

bioRxiv2023

Single molecule studies of the native hair cell mechanosensory transduction complex

Hearing and balance rely on the conversion of a mechanical stimulus into an electrical signal, a process known as mechanosensory transduction (MT). In vertebrates, this process is accomplished by an MT complex that is located in hair cells of the inner ear. While the past three decades of research have identified many subunits that are important for MT and revealed interactions between these subunits, the composition and organization of a functional complex remains unknown. The major challenge associated with studying the MT complex is its extremely low abundance in hair cells; current estimates of MT complex quantity range from 3-60 attomoles per cochlea or utricle, well below the detection limit of most biochemical assays that are used to characterize macromolecular complexes. Here we describe the optimization of two single molecule assays, single molecule pull-down (SiMPull) and single molecule array (SiMoA), to study the composition and quantity of native mouse MT complexes. We demonstrate that these assays are capable of detecting and quantifying low attomoles of the native MT subunits protocadherin-15 (PCDH15) and lipoma HMGIC fusion partner-like protein 5 (LHFPL5). Our results illuminate the stoichiometry of PCDH15- and LHFPL5-containing complexes and establish SiMPull and SiMoA as productive methods for probing the abundance, composition, and arrangement of subunits in the native MT complex. Impact StatementIn the present work, the authors develop and employ single molecule methods to detect, characterize, and quantitate attomole quantities of the hair cell mechanosensory transduction complex.

biochemistry↗

bioRxiv2023

Rapid long-distance migration of RPA on single stranded DNA occurs through intersegmental transfer utilizing multivalent interactions

Replication Protein A (RPA) is a single stranded DNA (ssDNA) binding protein that coordinates diverse DNA metabolic processes including DNA replication, repair, and recombination. RPA is a heterotrimeric protein with six functional oligosaccharide/oligonucleotide (OB) domains and flexible linkers. Flexibility enables RPA to adopt multiple configurations and is thought to modulate its function. Here, using single molecule confocal fluorescence microscopy combined with optical tweezers and coarse-grained molecular dynamics simulations, we investigated the diffusional migration of single RPA molecules on ssDNA under tension. The diffusion coefficient D is the highest (20,000 nucleotides2/s) at 3 pN tension and in 100 mM KCl and markedly decreases when tension or salt concentration increases. We attribute the tension effect to intersegmental transfer which is hindered by DNA stretching and the salt effect to an increase in binding site size and interaction energy of RPA-ssDNA. Our integrative study allowed us to estimate the size and frequency of intersegmental transfer events that occur through transient bridging of distant sites on DNA by multiple binding sites on RPA. Interestingly, deletion of RPA trimeric core still allowed significant ssDNA binding although the reduced contact area made RPA 15-fold more mobile. Finally, we characterized the effect of RPA crowding on RPA migration. These findings reveal how the high affinity RPA-ssDNA interactions are remodeled to yield access, a key step in several DNA metabolic processes. SignificanceReplication Protein A (RPA) binds to the exposed single stranded DNA (ssDNA) during DNA metabolism. RPA dynamics are essential to reposition RPA on ssDNA and recruit downstream proteins at the bound site. Here in this work, we perform a detailed biophysical study on dynamics of yeast RPA on ssDNA. We show that RPA can diffuse on ssDNA and is affected by tension and salt. Our observations are best explained by the intersegmental transfer model where RPA can transiently bridge two distant DNA segments for its migration over long distances. We further dissect the contributions of the trimerization core of RPA and other adjacent RPA molecules on RPA migration. This study provides detailed experimental and computational insights into RPA dynamics on ssDNA.

biophysics↗

bioRxiv2023

Electrostatic encoding of genome organization principles within single native nucleosomes

The eukaryotic genome, first packed into nucleosomes of about 150 bp around the histone core, is organized into euchromatin and heterochromatin, corresponding to the A and B compartments, respectively. Here, we asked if individual nucleosomes in vivo know where to go. That is, do mono-nucleosomes by themselves contain A/B compartment information, associated with transcription activity, in their biophysical properties? We purified native mono-nucleosomes to high monodispersity and used physiological concentrations of biological polyamines to determine their condensability. The chromosomal regions known to partition into A compartments have low condensability and vice versa. In silico chromatin polymer simulations using condensability as the only input showed that biophysical information needed to form compartments is all contained in single native nucleosomes and no other factors are needed. Condensability is also strongly anticorrelated with gene expression, and especially so near the promoter region and in a cell type dependent manner. Therefore, individual nucleosomes in the promoter know whether the gene is on or off, and that information is contained in their biophysical properties. Comparison with genetic and epigenetic features suggest that nucleosome condensability is a very meaningful axis onto which to project the high dimensional cellular chromatin state. Analysis of condensability using various condensing agents including those that are protein-based suggests that genome organization principle encoded into individual nucleosomes is electrostatic in nature. Polyamine depletion in mouse T cells, by either knocking out ornithine decarboxylase (ODC) or inhibiting ODC, results in hyperpolarized condensability, suggesting that when cells cannot rely on polyamines to translate biophysical properties of nucleosomes to control gene expression and 3D genome organization, they accentuate condensability contrast, which may explain dysfunction known to occur with polyamine deficiency.

biophysics↗

bioRxiv2023

Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis

Dynamin 1 (Dyn1) has two major splice variants, xA and xB, with unique C-terminal extensions of 20 and 7 amino acids, respectively. Of these, only Dyn1xA is enriched at endocytic zones and accelerates vesicle fission during ultrafast endocytosis. Here, we report that the long tail variant, Dyn1xA, achieves this localization by preferentially binding to Endophilin A through a newly defined Class II binding site overlapping with its extension, at a site spanning the splice boundary. Endophilin binds this site at higher affinity than the previously reported site, and this affinity is determined by amino acids outside the binding sites acting as long distance elements within the xA tail. Their interaction is regulated by the phosphorylation state of two serine residues specific to the xA variant. Dyn1xA and Endophilin colocalize in patches near the active zone of synapses. Mutations selectively disrupting Endophilin binding to the long extension cause Dyn1xA mislocalization along axons. In these mutants, endocytic pits are stalled on the plasma membrane during ultrafast endocytosis. These data suggest that the specificity for ultrafast endocytosis is defined by the phospho-regulated interaction of Endophilin A through a newly identified site of Dyn1xAs long tail.

cell biology↗

bioRxiv2023

GAGA Factor Overcomes 1D Diffusion Barrier by 3D Diffusion in Search of Nucleosomal Targets

To elucidate how eukaryotic sequence-specific transcription factors (TFs) search for gene targets on chromatin, we used multi-color smFRET and single-particle imaging to track the diffusion of purified GAGA-Associated Factor (GAF) on DNA and nucleosomes. Monomeric GAF DNA-binding domain (DBD) bearing one zinc finger finds its cognate site by 1D or 3D diffusion on bare DNA and rapidly slides back-and-forth between naturally clustered motifs for seconds before escape. Multimeric, full-length GAF also finds clustered motifs on DNA by 1D-3D diffusion, but remains locked on target for longer periods. Nucleosome architecture effectively blocks GAF-DBD 1D-sliding into the histone core but favors retention of GAF-DBD when targeting solvent-exposed sites by 3D-diffusion. Despite the occlusive power of nucleosomes, 1D-3D facilitated diffusion enables GAF to effectively search for clustered cognate motifs in chromatin, providing a mechanism for navigation to nucleosome and nucleosome-free sites by a member of the largest TF family.

biophysics↗

bioRxiv2023

Dynamic 1D Search and Processive Nucleosome Translocations by RSC and ISW2 Chromatin Remodelers

Eukaryotic gene expression is linked to chromatin structure and nucleosome positioning by ATP-dependent chromatin remodelers that establish and maintain nucleosome-depleted regions (NDRs) near transcription start-sites. Conserved yeast RSC and ISW2 remodelers exert antagonistic effects on nucleosomes flanking NDRs, but the temporal dynamics of remodeler search, engagement and directional nucleosome mobilization for promoter accessibility are unknown. Using optical tweezers and 2-color single-particle imaging, we investigated the Brownian diffusion of RSC and ISW2 on free DNA and sparse nucleosome arrays. RSC and ISW2 rapidly scan DNA by one-dimensional hopping and sliding respectively, with dynamic collisions between remodelers followed by recoil or apparent co-diffusion. Static nucleosomes block remodeler diffusion resulting in remodeler recoil or sequestration. Remarkably, both RSC and ISW2 use ATP hydrolysis to translocate mono-nucleosomes processively at [~]30 bp/sec on extended linear DNA under tension. Processivity and opposing push-pull directionalities of nucleosome translocation shown by RSC and ISW2 shape the distinctive landscape of promoter chromatin.

biophysics↗

bioRxiv2023

BuGZ exhibits guanine nucleotide exchange factor toward tubulin

- and {beta}-tubulin form heterodimers, with GTPase activity, that assemble into microtubules. Like other GTPases, the nucleotide-bound state of tubulin heterodimers controls whether the molecules are in a biologically active or inactive state. While -tubulin in the heterodimer is constitutively bound to GTP, {beta}-tubulin can be bound to either GDP (GDP-tubulin) or GTP (GTP-tubulin). GTP-tubulin hydrolyzes its GTP to GDP following assembly into a microtubule and, upon disassembly, must exchange its bound GDP for GTP to participate in subsequent microtubule polymerization. Tubulin dimers have been shown to exhibit rapid intrinsic nucleotide exchange in vitro, leading to a commonly accepted belief that a tubulin guanine nucleotide exchange factor (GEF) may be unnecessary in cells. Here, we use quantitative binding assays to show that BuGZ, a spindle assembly factor, binds tightly to GDP-tubulin, less tightly to GTP-tubulin, and weakly to microtubules. We further show that BuGZ promotes the incorporation of GTP into tubulin using a nucleotide exchange assay. The discovery of a tubulin GEF suggests a mechanism that may aid rapid microtubule assembly dynamics in cells.

biochemistry↗

bioRxiv2023

Fluorescence lifetime analysis of smFRET with contribution of PIFE on donor and acceptor

Single-molecule fluorescence resonance energy transfer (FRET) is a powerful technique based on dipole-dipole interaction between donor and acceptor fluorophores to observe inter- and intra-molecular dynamics in realtime with sensitivity to macro-molecular distances ([~] 2.5-10 nm). That said, some fluorophores have an inherent characteristic known as protein induced fluorescence enhancement (PIFE). PIFE is a photo-physical feature of dyes undergoing cis-trans transitions and occurs for protein-dye interactions closer than 3 nm. Here, the challenge is uncoupling the PIFE effect in the FRET data. Ignoring the PIFE effect in the analysis of the FRET data may lead to misinterpretation of the system under investigation. As a solution to this problem, we develop a computational framework based on Bayesian statistics to analyze the fluorescence lifetime signals of the donor and acceptor channels which allows us to uncouple the PIFE effects from the FRET. Our framework can extract any changes in the FRET efficiency simultaneously with any changes in the fluorescence lifetimes of the donor and acceptor due to the PIFE effect. In addition, our framework can provide other parameters, such as the donor and acceptor excitation rates, background photon rates, and detectors cross-talk ratios. Our framework extracts all these parameters by analyzing a single photon arrival time trace with only a few thousand photons.

biophysics↗

bioRxiv2023

Linking folding dynamics and function of SAM/SAH riboswitches at the single molecule level

Riboswitches are found in the 5-UTR of many bacterial mRNAs. They function as cisacting regulatory elements that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes of this translational switch alter ribosome accessibility. The folding of the riboswitch is highly heterogenous, indicating a complex and rugged conformational landscape that enables sampling of the ligand-bound conformation even in the absence of the ligand. Upon ligand binding, the landscape shifts towards the ligand-bound conformation. Mutations at key stabilizing structures alter the ligand-free folding behavior and decrease ligand responsiveness. We also explored translational regulation through folding kinetics by utilizing short oligonucleotides to probe the accessibility of the Shine-Dalgarno sequence within the riboswitch. Additionally, we employed a helicase-based vectorial folding assay to simulate co-transcriptional folding. We find that a competition between ligand binding and ribosome binding is fined tuned via the kinetics of folding. During transcription, the riboswitch takes minutes before reaching equilibrated conformations, and such slow equilibration decreases the effective ligand affinity. Overall, our data demonstrate the significance of conformational polymorphism in RNA function, emphasizing the utilization of complex folding landscapes for regulating ribosome accessibility through ligand induction. Furthermore, we provide direct evidence on how folding kinetics modulate this regulation process.

biophysics↗

bioRxiv2023

Massively parallel single molecule tracking of sequence-dependent DNA mismatch repair in vivo

Whether due to mutagens or replication errors, DNA mismatches arise spontaneously in vivo. Unrepaired mismatches are sources of genetic variation and point mutations which can alter cellular phenotype and cause dysfunction, diseases, and cancer. To understand how diverse mismatches in various sequence contexts are recognized and repaired, we developed a high-throughput sequencing-based approach to track single mismatch repair outcomes in vivo and determined the mismatch repair efficiencies of 5682 distinct singly mispaired sequences in E. coli. We found that CC mismatches are always poorly repaired, whereas local sequence context is a strong determinant of the hypervariable repair efficiency of TT, AG, and CT mismatches. Single molecule FRET analysis of MutS interactions with mismatched DNA showed that well-repaired mismatches have a higher effective rate of sliding clamp formation. The hypervariable repair of TT mismatches can cause selectively enhanced mutability if a failure to repair would result in synonymous codon change or a conservative amino acid change. Sequence-dependent repair efficiency in E. coli can explain the patterns of substitution mutations in mismatch repair-deficient tumors, human cells, and C. elegans. Comparison to biophysical and biochemical analyses indicate that DNA physics is the primary determinant of repair efficiency by its impact on the mismatch recognition by MutS.

biophysics↗

bioRxiv2023

Dimensional Reduction for Single Molecule Imaging of DNA and Nucleosome Condensation by Polyamines, HP1α and Ki-67

Macromolecules organize themselves into discrete membrane-less compartments. Mounting evidence has suggested that nucleosomes as well as DNA itself can undergo clustering or condensation to regulate genomic activity. Current in vitro condensation studies provide insight into the physical properties of condensates, such as surface tension and diffusion. However, such studies lack the resolution needed for complex kinetic studies of multicomponent condensation. Here, we use a supported lipid bilayer platform in tandem with total internal reflection microscopy to observe the 2-dimensional movement of DNA and nucleosomes at the single-molecule resolution. This dimensional reduction from 3-dimensional studies allows us to observe the initial condensation events and dissolution of these early condensates in the presence of physiological condensing agents. Using polyamines, we observed that the initial condensation happens on a timescale of minutes while dissolution occurs within seconds upon charge inversion. Polyamine valency, DNA length and GC content affect threshold polyamine concentration for condensation. Protein-based nucleosome condensing agents, HP1 and Ki-67, have much lower threshold concentration for condensation than charge-based condensing agents, with Ki-67 being the most effective as low as 100 pM for nucleosome condensation. In addition, we did not observe condensate dissolution even at the highest concentrations of HP1 and Ki-67 tested. We also introduce a two-color imaging scheme where nucleosomes of high density labeled in one color is used to demarcate condensate boundaries and identical nucleosomes of another color at low density can be tracked relative to the boundaries after Ki-67 mediated condensation. Our platform should enable the ultimate resolution of single molecules in condensation dynamics studies of chromatin components under defined physicochemical conditions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/522433v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1ed0cc9org.highwire.dtl.DTLVardef@1e26b5dorg.highwire.dtl.DTLVardef@1f6e73corg.highwire.dtl.DTLVardef@c7227b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice

The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5' strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site--a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate the sites of MRN-dependent processing by isolating and sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated with highest efficiency when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show much greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that Mre11 and DNA-PK both bind to DNA ends before release of DNA-PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.

molecular biology↗

bioRxiv2022

Sniper2L, a high-fidelity Cas9 variant with high activity

Although several high-fidelity SpCas9 variants that have reduced activities at mismatched target sequences have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. Here, we developed an improved version of Sniper-Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. We evaluated Sniper2L activities at a large number of target sequences, and developed DeepSniper, a deep-learning model that can predict the activity of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. We envision that Sniper2L will be useful when efficient and specific genome editing is required.

bioengineering↗