Biophysics Korea-Europe 2023

R-loop formed at DNA double-strand breaks plays an essential role in DNA repair, but its formation mechanisms remain elusive. We study co-transcriptional R-loop formation using in vitro single-molecule transcription assays and find that Escherichia coli RNA polymerase is an efficient DNA damage sensor that initiates R-loop formation at specific DNA lesions such as double-strand breaks, inter-strand crosslinking, and G-quadruplexes in the nontemplate strand. R-loop is extended mainly towards upstream from the RNA·DNA hybrid in the transcriptional bubble, and the extended R-loop hinders the ongoing and next-round transcriptions.

In this presentation, we will report our research progress on the graphene NEMS based nanomechanical resonator for a single molecule protein sequencing (SMPS). Current SMPS methods (based on the mass spectroscopy) suffer from the limitation that they fail to recognize minor species. Our idea for protein sequencing is to identify proteins at the ultimate sensitivity (one molecule) by using a nanomechanical resonator-based mass sensors. Non-linear mechanical resonance behavior was utilized to differentiate a small amount of frequency change by measuring the amplitude variation of graphene resonator. We also have improved the mass sensing resolution of graphene nanomechanical mass sensor by applying Joule-heating process, which provides a clean graphene membrane surface. We characterized the sensor performances and found a mass-resolution that is sufficient to weigh very small particles, like large proteins and protein complexes, with potential applications in the fields of nanobiology and medicine. In addition, we found out that the limit of mass change detection using GMS can be improved by using applied machine learning. Among several trials with various algorithm, 2D-CNN (convolutional neural networks) learning-based classification shows the best performance so that the mass detection signal can be recognized in noisy environment where the noise level is even 4 times higher than the signal level.

We observed that diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), induces mitotic arrest with a half-maximal effective concentration of 170 μM and cell death with a half-maximal lethal dose of 200 μM during 18-h incubation in HeLa cells. Cellular microtubule imaging and in vitro tubulin polymerization assays demonstrated that treatment with diclofenac elicits microtubule destabilization. Autophagy relies on microtubule-mediated transport and the fusion of autophagic vesicles. We observed that diclofenac inhibits both phagophore movement, an early step of autophagy, and the fusion of autophagosomes and lysosomes, a late step of autophagy. Diclofenac also induces the fragmentation of mitochondria and the Golgi during cell death. We found that diclofenac induces cell death further in combination with 5-fuorouracil, a DNA replication inhibitor than in single treatment in cancer cells. Pancreatic cancer cells, which have high basal autophagy, are particularly sensitive to cell death by diclofenac. Our study suggests that diclofenac may be a candidate therapeutic drug in certain type of cancers by inhibiting microtubule- mediated cellular events in combination with clinically utilized nucleoside metabolic inhibitors, including 5-fluorouracil, to block cancer cell proliferation. Mapping of spatiotemporal distribution and structural change of biomolecules including proteins is important to understand physiology inside cells and organism. Molecular imaging is one of best technology to satisfy this demand. Establishment of public data archives for bioimaging resources recently draws basic and clinical researchers’ attention. Data archives should be well organized to make these data findable, accessible, interoperable and reusable. Bioimaging data quality leading center has roles to distribute standard operating procedure (SOP) for production and analysis of bioimage metadata and connect different datasets on basis of common standardized elements to accessible and reusable to the scientific community as well as image analysis program developers.

The oxidative base damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is a highly mutagenic lesion because replicative DNA polymerases insert adenine (A) opposite 8-oxoG. In mammalian cells, the removal of A incorporated across from 8-oxoG is mediated by the glycosylase MUTYH during base excision repair (BER). After A excision, MUTYH binds avidly to the abasic site and is thus product inhibited. We have previously reported that UV-DDB plays a non-canonical role in BER during the removal of 8-oxoG by 8-oxoG glycosylase, OGG1 and presented preliminary data that UV-DDB can also increase MUTYH activity. In this present study we examine the mechanism of how UV-DDB stimulates MUTYH. Bulk kinetic assays show that UV-DDB can stimulate the turnover rate of MUTYH excision of A across from 8-oxoG by 4–5-fold. Electrophoretic mobility shift assays and atomic force microscopy suggest transient complex formation between MUTYH and UV-DDB, which displaces MUTYH from abasic sites. Using single molecule fluorescence analysis of MUTYH bound to abasic sites, we show that UV-DDB interacts directly with MUTYH and increases the mobility and dissociation rate of MUTYH. UV-DDB decreases MUTYH half-life on abasic sites in DNA from 8800 to 590 seconds. Together these data suggest that UV-DDB facilitates productive turnover of MUTYH at abasic sites during 8-oxoG:A repair.

Recent advances in single-molecule fluorescence microscope techniques have allowed single-molecule sensitivity to probe various protein-DNA interactions, their structural changes, and fundamental cellular processes in a living cell. Transcription, a process of mRNA generation by RNA polymerase (RNAP), is highly coupled with translation by the ribosome in bacteria. The effect of the transcription-translation coupling on the transcriptional dynamics and the localization of genes in a living cell is poorly understood. We directly observe the dynamics of transcription and the movement of the subcellular localization of genes actively transcribed by RNAP in living cells at the sub-diffraction limit resolution. The subcellular localizations of the non-membrane protein’ genes, actively transcribed by RNAPs, move toward outside nucleoid or to the plasma membrane by the effect of translation by ribosome. Not only for gene movement but translation also significantly affects mRNA generation. We proposed a new mechanism for controlling mRNA expression levels by translation. Our observation will provide new insight into the role of the coupling between transcription and translation on the effective expression of genes in E. coli.

Infectious diseases are caused by bacteria, viruses, fungi or parasites, and human genetic diseases are caused by mutations in one or more human genes. For instance, the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces Coronavirus disease 2019 (Covid-19) and point mutations in EGFR and KRAS genes are the most frequently mutated genes in lung cancer. Therefore, the ultrasensitive detection of foreign genes and DNA point mutations in human genes is highly required for diagnosis of infectious diseases and human genetic diseases at their early stage. Here, we suggest highly sensitive techniques to quantify DNA and a point mutation in DNA by combining the clustered regularly interspaced short palindromic repeats (CRISPR) system and single-molecule fluorescence detection. With our CRISPR-based single-molecule detection platform, we could manage to detect DNA and a DNA point mutation in a highly quantitative manner without employing any DNA amplification methods. We applied it to detect the amount of SARS-CoV-2 E gene and the fraction of EGFR c.2573 T>G mutation that is a genetic marker of lung cancer, and successfully quantified the SARS-CoV-2 at the subpicomolar level and the fraction of the point mutation down to ~ 0.5 % of minor allele.

Single-molecule fluorescence resonance energy transfer (smFRET) has been an indispensable tool to probe the structure and dynamics of biomolecules on the nanometer scale. The intrinsic low-throughput nature of single molecule detection has been, however, a hurdle for the technique to be used to study a large pool of samples. Here, I present a highly multiplexed smFRET approach by combining the conventional smFRET measurement with a next generation sequencing method. A library of DNA molecules, carrying thousands of different sequences of interest, are prepared and measured in a one pot recipe. By using this high-throughput approach, the sequence-dependent structural heterogeneity of single stranded DNA in various native aqueous buffer conditions are experimentally determined.

Label-free imaging of cellular structures and dynamics

Seok-Cheol Hong (Korea University)

Imaging a cell in its natural state is the first step towards understanding its biological behaviors and functions. Since simple light scattering, on which most of optical microscopy techniques are based, lacks chemical specificity and selectivity, fluorescence microscopy has been a workhorse to reveal biological secrets. Due to drawbacks such as limited SNR, photobleaching/blinking and need for labeling, however, non-fluorescent imaging techniques have long been dreamed of as an alternative and complementary tool. Recently, scattering-based imaging techniques are rejuvenated to circumvent the aforementioned limitations with their superb sensitivity, simplicity, and versatility and to capture previously inaccessible dynamic features of live cells. In this short presentation, I will present our recent achievements along this direction, highlighting label-free imaging of cellular complexes and their dynamic motions. 

Tunneling nanotubes (TNT) have been identified as a novel channel for long-distance communication between cells, connecting cells across extended distances of up to several cell diameters. However, it is unclear how such a fine structure can expand and remain robust for several hours. We examined the spatiotemporal processes of TNT formation using superresolution microscopy, which originated from filopodial bridges (two-filopodia complex: DFB). We discovered that the helical structures of DFB play an important role in the transition of DFB to TNT. TNT's end contacts the paired cell body via cadherin-cadherin adhesion, which tightly connects two cells. Torsional energy in DFBs accumulated by helical deformation destroys cadherin-cadherin interactions between two filopodia. Mechanical investigations and computer-aided simulations strongly imply that the shift from DFB to SFB is possible. We confirmed that cellular components could be transferred between cells via gap-junction molecules on closed-end TNT. Our investigation uncovered mechanistic issues in the TNT formation's battle lines 

The rise of super-resolution microscopy (SRM, Research field of the Nobel Prize in Chemistry 2014) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for single-molecule imaging. The new opportunities afforded by SRM have hence motivated extensive new research, providing multidimensional, multi-scale, and corroborated information about a system regarding morphology, functionality, dynamics, cellular context, and chemical composition. In this presentation, I will talk about our technology development and recent applications of SRM technique. Using this approach, we investigated the ultrastructural changes of platelets during the platelet release and activation as well as the extracellular vesicle biogenesis in gram-positive bacteria. These findings highlight the concerted ultrastructural reorganization and relative arrangements of various organelles upon these biological processes and call for a reassessment of previously unresolved complex and multi-factorial biological processes.

Phase separations involving biomolecular liquids are emerging as a fundamental mechanism of intracellular organization. Liquid-liquid phase separation (LLPS) of biomolecules compartmentalizes cellular contents into multiple coexisting condensates to facilitate spatio-temporal regulation of biological processes ranging from gene expression to cell signaling. In this talk, I will discuss our recent efforts to engineer DNA-based synthetic condensates of which the organization and function can be programmed at the sequence level. Inspired by the way intracellular condensates are organized, we control the assembly and composition of condensates through the fine tuning of individual intermolecular interactions. We demonstrate that DNA-based molecular computation can be drastically accelerated by coupling the reaction components into condensates. The additional layer of phase programmability provided by our synthetic condensate platforms will be useful to build advanced artificial systems with novel functionalities. 

DNA damage that is induced by endogenous or environmental factors threatens genomic integrity and stability, causing malignant diseases. To protect DNA from damaging agents, different repair mechanisms have evolved according to the types of DNA damage. Eukaryotic DNA is packaged into the small nucleus by forming chromatin, which is a higher-order structure. Chromatin structure alters according to cell cycle and external stimuli. The chromatin dynamics highly influences DNA metabolism such as DNA repair. To date, DNA repair and chromatin dynamics have been separately studied. How repair enzymes find and remove DNA lesions in the chromatinized DNA and how chromatin organization is restored after repair remain poorly understood. We systematically investigate how human repair proteins mend impaired DNA in the chromatin context using a novel single-molecule imaging technique, DNA curtain. We reveal the damage search mechanism of human NER protein XPC-RAD23B and R-loop sensing mechanism of TonEBP. For the chromatin dynamics, we determine the molecular role of bromodomain-containing AAA+ ATPases in nucleosome assembly. Our research will provide insight into DNA metabolism in chromatin by bridging DNA damage repair and chromatin dynamics.

Direct visualization of the genomic elements in living cells is required to explore the relationship between the dynamic organization of chromatin and its functional roles. Using a CRISPR-based genome imaging system, we visualized large centromeric and pericentromeric domains in live cells. CRISPR labeling of a large domain with high density induced DNA damage response. As a result, in S phase, the domain expanded and exhibited highly extended fiber-like structure that reached out over several microns with complex features such as branching, bridging, and looping. The labeled domain colocalized with repair pathway proteins such as γH2AX, pATM, and pCHK2. PCNA clusters were found to localize where the chromatin fibers stem from the domain body. When dCas9 was fused to 53BP1, the fiber extension as well as the domain expansion disappeared, suggesting that the fusion forced the damaged region to follow the NHEJ pathway by preventing BRCA1 from knocking off 53BP1. This in turn implies that the expansion and extension of the damaged domain were the result of homology-directed repair process. This finding suggests an imaging-based system for the study of long-range chromatin dynamics in the homology-directed repair of DNA double-strand breaks in live cells.

Structural maintenance of chromosome (SMC) protein complexes are key proteins for genome organization by extruding DNA loops. However, it is not unclear whether only DNA loop extrusion can build the genome structures at a physiologically relevant condition. Here, we show that yeast cohesin, human cohesin and yeast condensin SMC complexes exhibit pronounced clustering on DNA, with all the hallmarks of biomolecular condensation (1). DNA-SMC clusters exhibit liquid-like behavior, showing fusion of clusters, rapid fluorescence recovery after photobleaching and exchange of SMC complexes with the environment. Strikingly, the in vitro clustering is DNA length dependent, as both yeast cohesin and human cohesin form clusters only on DNA exceeding 3 kilo–base pairs. We discuss how bridging-induced phase separation (BIPS), a previously unobserved type of biological condensation, can explain the DNA-protein clustering through DNA-protein-DNA bridges. We confirm that, in yeast cells in vivo, a fraction of cohesin associates with chromatin in a manner consistent with BIPS. BIPS likely is a universal phenomenon among SMC proteins. Biomolecular condensation by SMC proteins constitutes a new basic principle by which SMC complexes direct genome organization. 

DNA is a long polymeric substrate that provides specific binding sites for many proteins in cell nucleus. Both diffusion and directed translocation play an essential role for protein-DNA interactions in gene regulation processes but it is still not fully understood how specific proteins move and search their target sites in the myriad of DNA sequence repertoires. We use λ-exonuclease as a model of nucleic acid-molecular motors that processively translocates along the DNA. Here, we combined single molecule FRET and molecular dynamics (MD) simulation to examine how the dynamic interaction transmits into overall enzymatic activity. Transient coupling between λ-exonuclease and its substrate DNA significantly alters its translocation by a factor of ~30 due to chemical friction between a positive reside (ARG45) of the protein and electrostatic potential (EP) along the minor groove of DNA. Repulsive interaction gives rise to futile slippage events whereas attractive coupling between ARG45 and adenines at the minor grooves provides chemical ratcheting for unidirectional translocation, preventing diffusive backtracking by electrostatic friction. We propose an anti-friction-based ratchet for processive translocation. Our study provides new insights into not only interplay between dynamic chemophysical interaction and enzyme activity but also a role of the minor groove in regulating enzymatic activity based on DNA sequences.

Despite advances in resolving structures of multi-pass membrane proteins, little is known about the native folding pathways of these complex structures. Using single-molecule magnetic tweezers, we report a complete folding pathway of purified human glucose transporter 3 (GLUT3) reconstituted within lipid bilayers composed of synthetic lipids and detergents. The N-terminal major facilitator superfamily (MFS) fold strictly forms first, serving as structural templates for its C-terminal counterpart that defines most of the glucose binding site. Our data further reveal folding pathways for individual MFS folds, where polar residues comprising the membrane-embedded conduit for glucose molecules present major folding challenges. The ER membrane protein complex facilitates insertion of more hydrophilic TMHs, thrusting GLUT3’s microstate sampling toward folded structures. Final assembly between the N- and C-terminal MFS folds depends on specific lipids that ease desolvation of lipid shells surrounding the domain interfaces. Sequence analysis suggests that this asymmetric folding propensity across the N- and C-terminal MFS folds may prevail for metazoan sugar porters, revealing evolutionary conflicts between foldability and functionality faced by many multi-pass membrane proteins.

Structural Maintenance of Chromosomes (SMC) proteins like cohesin and condensin spatially organize chromosomes by extruding DNA into large loops. Using single-molecule assays, we provided unambiguous evidence for loop extrusion by directly visualizing the processive extension of DNA loops by SMCs in real-time. In recent extensions of this work, we showed that SMC proteins can bypass huge roadblocks of bound proteins on DNA, and we made progress in understanding the molecular mechanism that underlies this remarkable new class of DNA-translocating motor proteins.

Eucaryotic DNA is organized in chromatin, the assembly of strings of an octamer of histone proteins and ~150 DNA, realizing not only a strong compaction but also a means to regulate transcription and other activities on DNA. However, the structure of chromatin has been illusive for a long time, and recent studies even suggest a largely disordered organization at the molecular scale. Here, we use a combination of single-molecule biophysical tools and statistical physics methods, to get a better physical grip on how our genome is organized. I will discuss our recent findings on how the structure of chromatin depends on the underlying sequence and quantify how higher order folding regulates the accessibility of our DNA. Statistical physics can largely explain the sequence dependence of nucleosome positioning and chromatin folding. Using single-pair FRET and magnetic tweezers-based force spectroscopy, we showed how embedding of a nucleosome in a fiber affects the accessibility of nucleosomal DNA. Overall, the general picture is that DNA sequence directs nucleosome positions and that nucleosome positions, in combination with the sequence of linker DNA drive alternative higher order structures.

Eukaryotic organisms have evolved motor proteins to drive long-distance transport. Single-molecule methods, including fluorescence microscopy have provided important new insights into the molecular mechanisms of motor proteins like kinesin and dynein. In the past, most of these studies were in vitro, now motor proteins are studied increasing in their cellular environment. In our cells, motor proteins do not work on their own: cargoes are often transported by multiple motors often of opposite directionality. In addition, our cells are a very crowded environment, with many proteins bound to the motors’ tracks, which might hamper their motion and could lead to ‘traffic jams’.

To study such problems, we have focused on a specific transport mechanism, intraflagellar transport (IFT), which is essential for the assembly and maintenance of cilia. In particular, we study IFT in the chemosensory cilia of the nematode C. elegans. In these worms, IFT is driven by groups of tens of three different motor proteins: 2 different kinesins drive transport of cargo trains from base of the cilium to tip, while IFT dynein drives transport back to the base. We use live wide-field fluorescence microscopy on transgenic, fluorescent worms to unravel the molecular mechanism of IFT. Our fluorescence and image analysis approaches allow us to visualize, track and quantify trains of IFT components moving together as well as individual motors or IFT proteins. Together, bulk and single-molecule data recorded in living worms provide new, deep insights into the mechanisms of motor cooperation.