Publications by authors named "Sungchul Hohng"

57 Publications

A Novel N-terminal Region to Chromodomain in CHD7 is Required for the Efficient Remodeling Activity.

J Mol Biol 2021 Jun 21;433(18):167114. Epub 2021 Jun 21.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), KAIST Institute of BioCentury, Daejeon 34141, Korea. Electronic address:

Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed biochemical and biophysical analysis on CHD7 chromatin remodeler and uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome and contains a high conserved arginine stretch, which is reminiscent of arginine anchor. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. Furthermore, smFRET analysis shows the mutations in the N-CRD causes the defects in remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities.
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http://dx.doi.org/10.1016/j.jmb.2021.167114DOI Listing
June 2021

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Elife 2021 03 29;10. Epub 2021 Mar 29.

Departments of Biology and Chemistry, Johannes Gutenberg University, Mainz, Germany.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.
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http://dx.doi.org/10.7554/eLife.60416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007216PMC
March 2021

Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription.

Int J Mol Sci 2021 Feb 27;22(5). Epub 2021 Feb 27.

Department of Physics and Astronomy, and Institute of Applied Physics, Seoul National University, Seoul 08826, Korea.

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.
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http://dx.doi.org/10.3390/ijms22052398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957599PMC
February 2021

Quantification of purified endogenous miRNAs with high sensitivity and specificity.

Nat Commun 2020 11 27;11(1):6033. Epub 2020 Nov 27.

Department of Physics and Astronomy, and Institute of Applied Physics, Seoul National University, Seoul, Republic of Korea.

MicroRNAs (miRNAs) are short (19-24 nt) non-coding RNAs that suppress the expression of protein coding genes at the post-transcriptional level. Differential expression profiles of miRNAs across a range of diseases have emerged as powerful biomarkers, making a reliable yet rapid profiling technique for miRNAs potentially essential in clinics. Here, we report an amplification-free multi-color single-molecule imaging technique that can profile purified endogenous miRNAs with high sensitivity, specificity, and reliability. Compared to previously reported techniques, our technique can discriminate single base mismatches and single-nucleotide 3'-tailing with low false positive rates regardless of their positions on miRNA. By preloading probes in Thermus thermophilus Argonaute (TtAgo), miRNAs detection speed is accelerated by more than 20 times. Finally, by utilizing the well-conserved linearity between single-molecule spot numbers and the target miRNA concentrations, the absolute average copy numbers of endogenous miRNA species in a single cell can be estimated. Thus our technique, Ago-FISH (Argonaute-based Fluorescence In Situ Hybridization), provides a reliable way to accurately profile various endogenous miRNAs on a single miRNA sensing chip.
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http://dx.doi.org/10.1038/s41467-020-19865-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699633PMC
November 2020

Yeast Chd1p Unwraps the Exit Side DNA upon ATP Binding to Facilitate the Nucleosome Translocation Occurring upon ATP Hydrolysis.

Biochemistry 2020 12 11;59(47):4481-4487. Epub 2020 Nov 11.

Department of Physics and Astronomy, Institute of Applied Physics, Seoul National University, Seoul 08826, Republic of Korea.

Chromodomain-helicase-DNA-binding protein 1 (CHD1) remodels chromatin by translocating nucleosomes along DNA, but its mechanism remains poorly understood. We use single-molecule fluorescence experiments to clarify the mechanism by which yeast CHD1 (Chd1p) remodels nucleosomes. We find that binding of ATP to Chd1p induces transient unwrapping of the DNA on the exit side of the nucleosome, facilitating nucleosome translocation. ATP hydrolysis is required to induce nucleosome translocation. The unwrapped DNA after translocation is then rewrapped after the release of the hydrolyzed nucleotide and phosphate, revealing that each step of the ATP hydrolysis cycle is responsible for a distinct step of nucleosome remodeling. These results show that Chd1p remodels nucleosomes via a mechanism that is unique among the other ATP-dependent chromatin remodelers.
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http://dx.doi.org/10.1021/acs.biochem.0c00747DOI Listing
December 2020

Single-molecule fluorescence studies on cotranscriptional G-quadruplex formation coupled with R-loop formation.

Nucleic Acids Res 2020 09;48(16):9195-9203

Department of Physics and Astronomy, and Institute of Applied Physics, Seoul National University, Seoul 08826, Republic of Korea.

G-quadruplex (GQ) is formed at various regions of DNA, including telomeres of chromosomes and regulatory regions of oncogenes. Since GQ is important in both gene regulation and genome instability, the biological and medical implications of this abnormal DNA structure have been intensively studied. Its formation mechanisms, however, are not clearly understood yet. We report single-molecule fluorescence experiments to monitor the cotranscriptional GQ formation coupled with R-loop formation using T7 RNA polymerase. The GQ is formed very rarely per single-round transcription. R-loop formation precedes and facilitates GQ formation. Once formed, some GQs are extremely stable, resistant even to RNase H treatment, and accumulate in multiple-round transcription conditions. On the other hand, GQ existing in the non-template strand promotes the R-loop formation in the next rounds of transcription. Our study clearly shows the existence of a positive feedback mechanism of GQ and R-loop formations, which may possibly contribute to gene regulation and genome instability.
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http://dx.doi.org/10.1093/nar/gkaa695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498336PMC
September 2020

Transcription reinitiation by recycling RNA polymerase that diffuses on DNA after releasing terminated RNA.

Nat Commun 2020 01 23;11(1):450. Epub 2020 Jan 23.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea.

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from the DNA template much more often than their concurrent dissociations in intrinsic termination of bacterial transcription. As termination is defined by the release of product RNA from the transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as recycling. During the recycling stage, post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on the DNA template for reinitiation most of the time.
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http://dx.doi.org/10.1038/s41467-019-14200-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978380PMC
January 2020

Single-Molecule FRET Assay for Studying Cotranscriptional RNA Folding.

Methods Mol Biol 2020 ;2106:271-282

Department of Physics and Astronomy, Seoul National University, Seoul, Republic of Korea.

Cotranscriptional RNA folding plays important roles in gene regulation steps such as splicing, transcription termination, and translation initiation. Progression of our understanding of cotranscriptional RNA folding mechanisms is still retarded by the lacking of experimental tools to study the kinetics of cotranscriptional RNA folding properly. In this chapter, we describe fluorescence resonance energy transfer (FRET) assay that enables the study of RNA cotranscriptional folding at the single-molecule level.
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http://dx.doi.org/10.1007/978-1-0716-0231-7_17DOI Listing
June 2021

ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

Nat Commun 2019 12 16;10(1):5718. Epub 2019 Dec 16.

Center for Genomic Integrity, Institute for Basic Science, Ulsan, Korea.

Maintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2'-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.
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http://dx.doi.org/10.1038/s41467-019-13667-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914801PMC
December 2019

Structural basis of recognition and destabilization of the histone H2B ubiquitinated nucleosome by the DOT1L histone H3 Lys79 methyltransferase.

Genes Dev 2019 06 28;33(11-12):620-625. Epub 2019 Mar 28.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea.

DOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B ubiquitination. Here, we present cryo-EM structures of DOT1L complexes with unmodified or H2B ubiquitinated nucleosomes, showing that DOT1L recognizes H2B ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L, respectively. Furthermore, the structures combined with single-molecule FRET experiments show that H2B ubiquitination enhances a noncatalytic function of the DOT1L-destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.
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http://dx.doi.org/10.1101/gad.323790.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546062PMC
June 2019

Accelerated FRET-PAINT microscopy.

Mol Brain 2018 11 22;11(1):70. Epub 2018 Nov 22.

Department of Physics and Astronomy, Seoul National University, Seoul, 08826, Republic of Korea.

Recent development of FRET-PAINT microscopy significantly improved the imaging speed of DNA-PAINT, the previously reported super-resolution fluorescence microscopy with no photobleaching problem. Here we try to achieve the ultimate speed limit of FRET-PAINT by optimizing the camera speed, dissociation rate of DNA probes, and bleed-through of the donor signal to the acceptor channel, and further increase the imaging speed of FRET-PAINT by 8-fold. Super-resolution imaging of COS-7 microtubules shows that high-quality 40-nm resolution images can be obtained in just tens of seconds.
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http://dx.doi.org/10.1186/s13041-018-0414-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249777PMC
November 2018

ATP Binding to Rad5 Initiates Replication Fork Reversal by Inducing the Unwinding of the Leading Arm and the Formation of the Holliday Junction.

Cell Rep 2018 05;23(6):1831-1839

Department of Physics and Astronomy, Institute of Applied Physics, National Center of Creative Research Initiatives, Seoul National University, Seoul 08826, Republic of Korea. Electronic address:

Replication fork reversal is one of the major pathways for reactivating stalled DNA replication. Many enzymes with replication fork reversal activity have DNA-unwinding activity as well, but none of the fork reversal enzymes in the SWI/SNF family shows a separate DNA-unwinding activity, raising the question of how they initiate the remodeling process. Here, we found ATP binding to Rad5 induces the unwinding of the leading arm of the replication fork and proximally positions the leading and lagging arms. This facilitates the spontaneous remodeling of the replication fork into a four-way junction. Once the four-way junction is formed, Rad5 migrates the four-way junction at a speed of 7.1 ± 0.14 nt/s. The 3' end anchoring of the leading arm by Rad5's HIRAN domain is critical for both branch migration and the recovery of the three-way junction, but not for the structural transition to the four-way junction.
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http://dx.doi.org/10.1016/j.celrep.2018.04.029DOI Listing
May 2018

Multiple RPAs make WRN syndrome protein a superhelicase.

Nucleic Acids Res 2018 05;46(9):4689-4698

Department of Physics and Astronomy, Institute of Applied Physics, National Center of Creative Research Initiatives, Seoul National University, Seoul, Republic of Korea.

RPA is known to stimulate the helicase activity of Werner syndrome protein (WRN), but the exact stimulation mechanism is not understood. We use single-molecule FRET and magnetic tweezers to investigate the helicase activity of WRN and its stimulation by RPA. We show that WRN alone is a weak helicase which repetitively unwind just a few tens of base pairs, but that binding of multiple RPAs to the enzyme converts WRN into a superhelicase that unidirectionally unwinds double-stranded DNA more than 1 kb. Our study provides a good case in which the activity and biological functions of the enzyme may be fundamentally altered by the binding of cofactors.
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http://dx.doi.org/10.1093/nar/gky272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961295PMC
May 2018

Superresolution fluorescence microscopy for 3D reconstruction of thick samples.

Mol Brain 2018 03 15;11(1):17. Epub 2018 Mar 15.

Department of Physics and Astronomy, Seoul National University, Seoul, Republic of Korea.

Three-dimensional (3D) reconstruction of thick samples using superresolution fluorescence microscopy remains challenging due to high level of background noise and fast photobleaching of fluorescence probes. We develop superresolution fluorescence microscopy that can reconstruct 3D structures of thick samples with both high localization accuracy and no photobleaching problem. The background noise is reduced by optically sectioning the sample using line-scan confocal microscopy, and the photobleaching problem is overcome by using the DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography). As demonstrations, we take 3D superresolution images of microtubules of a whole cell, and two-color 3D images of microtubules and mitochondria. We also present superresolution images of chemical synapse of a mouse brain section at different z-positions ranging from 0 μm to 100 μm.
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http://dx.doi.org/10.1186/s13041-018-0361-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856285PMC
March 2018

Accelerated super-resolution imaging with FRET-PAINT.

Mol Brain 2017 12 28;10(1):63. Epub 2017 Dec 28.

Department of Physics and Astronomy, Seoul National University, Seoul, 08826, Republic of Korea.

Super-resolution fluorescence microscopy in the current form is hard to be used to image the neural connectivity of thick tissue samples due to problems such as slow imaging speed, severe photobleaching of fluorescent probes, and high background noise. Recently developed DNA-PAINT solved the photobleaching problem, but its imaging speed is extremely low. We report accelerated super-resolution fluorescence microscopy named FRET-PAINT. Compared to conventional DNA-PAINT, the imaging speed of the microscopy increases up to ~30-fold. As demonstrations, we show that 25-50 second imaging time is long enough to provide super-resolution reconstruction of microtubules and mitochondria of COS-7 cells.
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http://dx.doi.org/10.1186/s13041-017-0344-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5747120PMC
December 2017

Single-molecule FRET studies on the cotranscriptional folding of a thiamine pyrophosphate riboswitch.

Proc Natl Acad Sci U S A 2018 01 26;115(2):331-336. Epub 2017 Dec 26.

Department of Physics and Astronomy, Seoul National University, Seoul 08826, Republic of Korea;

Because RNAs fold as they are being synthesized, their transcription rate can affect their folding. Here, we report the results of single-molecule fluorescence studies that characterize the ligand-dependent cotranscriptional folding of the riboswitch that regulates translation. We found that the riboswitch aptamer folds into the "off" conformation independent of its ligand, but switches to the "on" conformation during transcriptional pausing near the translational start codon. Ligand binding maintains the riboswitch in the off conformation during transcriptional pauses. We expect our assay will permit the controlled study of the two main physical mechanisms that regulate cotranscriptional folding: transcriptional pausing and transcriptional speed.
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http://dx.doi.org/10.1073/pnas.1712983115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777041PMC
January 2018

Extended depth of field for single biomolecule optical imaging-force spectroscopy.

Opt Express 2017 Dec;25(25):32189-32197

Real-time optical imaging combined with single-molecule manipulation broadens the horizons for acquiring information about the spatiotemporal localization and the mechanical details of target molecules. To obtain an optical signal outside the focal plane without unintended interruption of the force signal in single-molecule optical imaging-force spectroscopy, we developed an optical method to extend the depth of field in a high numerical aperture objective (≥ 1.2), required to visualize a single fluorophore. By axial scanning, using an electrically tunable lens with a fixed sample, we were successfully able to visualize the epidermal growth factor receptor (EGFR) moving along the three-dimensionally elongated filamentous actin bundles connecting cells (intercellular nanotube), while another EGFR on the intercellular nanotube was trapped by optical tweezers in living cells. Our approach is simple, fast and inexpensive, but it is powerful for imaging target molecules axially in single-molecule optical imaging-force spectroscopy.
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http://dx.doi.org/10.1364/OE.25.032189DOI Listing
December 2017

Increased PKMζ activity impedes lateral movement of GluA2-containing AMPA receptors.

Mol Brain 2017 Nov 29;10(1):56. Epub 2017 Nov 29.

Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, South Korea.

Protein kinase M zeta (PKMζ), a constitutively active, atypical protein kinase C isoform, maintains a high level of expression in the brain after the induction of learning and long-term potentiation (LTP). Further, its overexpression enhances long-term memory and LTP. Thus, multiple lines of evidence suggest a significant role for persistently elevated PKMζ levels in long-term memory. The molecular mechanisms of how synaptic properties are regulated by the increase in PKMζ, however, are still largely unknown. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) mediates most of the fast glutamatergic synaptic transmission in the brain and is known to be critical for the expression of synaptic plasticity and memory. Importance of AMPAR trafficking has been implicated in PKMζ-mediated cellular processes, but the detailed mechanisms, particularly in terms of regulation of AMPAR lateral movement, are not well understood. In the current study, using a single-molecule live imaging technique, we report that the overexpression of PKMζ in hippocampal neurons immobilized GluA2-containing AMPARs, highlighting a potential novel mechanism by which PKMζ may regulate memory and synaptic plasticity.
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http://dx.doi.org/10.1186/s13041-017-0334-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716381PMC
November 2017

NAP1L1 accelerates activation and decreases pausing to enhance nucleosome remodeling by CSB.

Nucleic Acids Res 2017 05;45(8):4696-4707

Department of Physics and Astronomy, Seoul National University, Seoul 08826, Republic of Korea.

Cockayne syndrome protein B (CSB) belongs to the SWI2/SNF2 ATP-dependent chromatin remodeler family, and CSB is the only ATP-dependent chromatin remodeler essential for transcription-coupled nucleotide excision DNA repair. CSB alone remodels nucleosomes ∼10-fold slower than the ACF remodeling complex. Strikingly, NAP1-like histone chaperones interact with CSB and greatly enhance CSB-mediated chromatin remodeling. While chromatin remodeling by CSB and NAP1-like proteins is crucial for efficient transcription-coupled DNA repair, the mechanism by which NAP1-like proteins enhance chromatin remodeling by CSB remains unknown. Here we studied CSB's DNA-binding and nucleosome-remodeling activities at the single molecule level in real time. We also determined how the NAP1L1 chaperone modulates these activities. We found that CSB interacts with DNA in two principle ways: by simple binding and a more complex association that involves gross DNA distortion. Remarkably, NAP1L1 suppresses both these interactions. Additionally, we demonstrate that nucleosome remodeling by CSB consists of three distinct phases: activation, translocation and pausing, similar to ACF. Importantly, we found that NAP1L1 promotes CSB-mediated remodeling by accelerating both activation and translocation. Additionally, NAP1L1 increases CSB processivity by decreasing the pausing probability during translocation. Our study, therefore, uncovers the different steps of CSB-mediated chromatin remodeling that can be regulated by NAP1L1.
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http://dx.doi.org/10.1093/nar/gkx188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5416873PMC
May 2017

AUF1 facilitates microRNA-mediated gene silencing.

Nucleic Acids Res 2017 Jun;45(10):6064-6073

Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.

Eukaryotic mRNA decay is tightly modulated by RNA-binding proteins (RBPs) and microRNAs (miRNAs). RBP AU-binding factor 1 (AUF1) has four isoforms resulting from alternative splicing and is critical for miRNA-mediated gene silencing with a distinct preference of target miRNAs. Previously, we have shown that AUF1 facilitates miRNA loading to Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex. Here, we further demonstrate that depletion of AUF1 abolishes the global interaction of miRNAs and AGO2. Single-molecule analysis revealed that AUF1 slowed down assembly of AGO2-let-7b-mRNA complex unexpectedly. However, target mRNAs recognized by both miRNA and AUF1 are less abundant upon AUF1 overexpression implying that AUF1 is a decay-promoting factor influencing multiple steps in AGO2-miRNA-mediated mRNA decay. Our findings indicate that AUF1 functions in promoting miRNA-mediated mRNA decay globally.
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http://dx.doi.org/10.1093/nar/gkx149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449627PMC
June 2017

Single-Molecule Fluorescence Energy Transfer Assays for the Characterization of Reaction Pathways of miRNA-Argonaute Complex.

Methods Mol Biol 2017 ;1517:305-315

Department of Physics and Astronomy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.

Argonaute proteins are key components of the microRNA-induced silencing complexes (miRISCs) that mediate the posttranscriptional gene silencing of microRNAs and small interfering RNA (siRNAs). The complex reaction mechanism of miRISC is expected to be characterized by tracing the reaction pathways of miRISC at the single-molecule level in real time. In this chapter, we describe single-molecule fluorescence resonance energy transfer (FRET) assays to observe the target binding and reaction pathways of miRISC composed of a recombinant Argonaute and a small RNA.
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http://dx.doi.org/10.1007/978-1-4939-6563-2_21DOI Listing
January 2018

Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase.

Structure 2016 08 14;24(8):1292-1300. Epub 2016 Jul 14.

Department of Physics and Astronomy, Seoul National University, Seoul 08826, Republic of Korea; Institute of Applied Physics, Seoul National University, Seoul 08826, Republic of Korea; National Center for Creative Research Initiatives, Seoul National University, Seoul 08826, Republic of Korea; Department of Biophysics and Chemical Biology, Seoul National University, Seoul 08826, Republic of Korea. Electronic address:

The reactivation of stalled DNA replication via fork regression invokes Holliday junction formation, branch migration, and the recovery of the replication fork after DNA repair or error-free DNA synthesis. The coordination mechanism for these DNA structural transitions by molecular motors, however, remains unclear. Here we perform single-molecule fluorescence experiments with Werner syndrome protein (WRN) and model replication forks. The Holliday junction is readily formed once the lagging arm is unwound, and migrated unidirectionally with 3.2 ± 0.03 bases/s velocity. The recovery of the replication fork was controlled by branch migration reversal of WRN, resulting in repetitive fork regression. The Holliday junction formation, branch migration, and migration direction reversal are all ATP dependent, revealing that WRN uses the energy of ATP hydrolysis to actively coordinate the structural transitions of DNA.
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http://dx.doi.org/10.1016/j.str.2016.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167498PMC
August 2016

Structure of Human DROSHA.

Cell 2016 Jan 31;164(1-2):81-90. Epub 2015 Dec 31.

Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea; School of Biological Sciences, Seoul National University, Seoul 08826, Korea. Electronic address:

MicroRNA maturation is initiated by RNase III DROSHA that cleaves the stem loop of primary microRNA. DROSHA functions together with its cofactor DGCR8 in a heterotrimeric complex known as Microprocessor. Here, we report the X-ray structure of DROSHA in complex with the C-terminal helix of DGCR8. We find that DROSHA contains two DGCR8-binding sites, one on each RNase III domain (RIIID), which mediate the assembly of Microprocessor. The overall structure of DROSHA is surprisingly similar to that of Dicer despite no sequence homology apart from the C-terminal part, suggesting that DROSHA may have evolved from a Dicer homolog. DROSHA exhibits unique features, including non-canonical zinc-finger motifs, a long insertion in the first RIIID, and the kinked link between Connector helix and RIIID, which explains the 11-bp-measuring "ruler" activity of DROSHA. Our study implicates the evolutionary origin of DROSHA and elucidates the molecular basis of Microprocessor assembly and primary microRNA processing.
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http://dx.doi.org/10.1016/j.cell.2015.12.019DOI Listing
January 2016

Single-molecule fluorescence measurements reveal the reaction mechanisms of the core RISC, composed of human Argonaute 2 and a guide RNA.

BMB Rep 2015 Dec;48(12):643-4

Department of Physics and Astronomy, Institute of Applied Physics, National Center of Creative Research Initiatives, and Department of Biophysics and Chemical Biology, Seoul National University, Seoul 08826, Korea.

In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791318PMC
http://dx.doi.org/10.5483/bmbrep.2015.48.12.235DOI Listing
December 2015

AUF1 promotes let-7b loading on Argonaute 2.

Genes Dev 2015 Aug;29(15):1599-604

Laboratory of Genetics, National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224, USA;

Eukaryotic gene expression is tightly regulated post-transcriptionally by RNA-binding proteins (RBPs) and microRNAs. The RBP AU-rich-binding factor 1 (AUF1) isoform p37 was found to have high affinity for the microRNA let-7b in vitro (Kd = ∼ 6 nM) in cells. Ribonucleoprotein immunoprecipitation, in vitro association, and single-molecule-binding analyses revealed that AUF1 promoted let-7b loading onto Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex (RISC). In turn, AGO2-let-7 triggered target mRNA decay. Our findings uncover a novel mechanism by which AUF1 binding and transfer of microRNA let-7 to AGO2 facilitates let-7-elicited gene silencing.
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http://dx.doi.org/10.1101/gad.263749.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536308PMC
August 2015

Human Argonaute 2 Has Diverse Reaction Pathways on Target RNAs.

Mol Cell 2015 Jul;59(1):117-24

Department of Physics and Astronomy, Seoul National University, Seoul 151-747, Republic of Korea; Institute of Applied Physics, Seoul National University, Seoul 151-747, Republic of Korea; National Center of Creative Research Initiatives, Seoul National University, Seoul 151-747, Republic of Korea; Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-747, Republic of Korea. Electronic address:

Argonaute is a key enzyme of various RNA silencing pathways. We use single-molecule fluorescence measurements to characterize the reaction mechanisms of the core-RISC (RNA-induced silencing complex) composed of human Argonaute 2 and a small RNA. We found that target binding of core-RISC starts at the seed region, resulting in four distinct reaction pathways: target cleavage, transient binding, stable binding, and Argonaute unloading. The target cleavage requires extensive sequence complementarity and dramatically accelerates core-RISC recycling. The stable binding of core-RISC is efficiently established with the seed match only, providing a potential explanation for the seed-match rule of miRNA (microRNA) target selection. Target cleavage on perfect-match targets sensitively depends on RNA sequences, providing an insight into designing more efficient siRNAs (small interfering RNAs).
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http://dx.doi.org/10.1016/j.molcel.2015.04.027DOI Listing
July 2015

Functional Anatomy of the Human Microprocessor.

Cell 2015 Jun 28;161(6):1374-87. Epub 2015 May 28.

Center for RNA Research, Institute for Basic Science, Seoul 151-742, Korea; School of Biological Sciences, Seoul National University, Seoul 151-742, Korea. Electronic address:

MicroRNA (miRNA) maturation is initiated by Microprocessor composed of RNase III DROSHA and its cofactor DGCR8, whose fidelity is critical for generation of functional miRNAs. To understand how Microprocessor recognizes pri-miRNAs, we here reconstitute human Microprocessor with purified recombinant proteins. We find that Microprocessor is an ∼364 kDa heterotrimeric complex of one DROSHA and two DGCR8 molecules. Together with a 23-amino acid peptide from DGCR8, DROSHA constitutes a minimal functional core. DROSHA serves as a "ruler" by measuring 11 bp from the basal ssRNA-dsRNA junction. DGCR8 interacts with the stem and apical elements through its dsRNA-binding domains and RNA-binding heme domain, respectively, allowing efficient and accurate processing. DROSHA and DGCR8, respectively, recognize the basal UG and apical UGU motifs, which ensure proper orientation of the complex. These findings clarify controversies over the action mechanism of DROSHA and allow us to build a general model for pri-miRNA processing.
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http://dx.doi.org/10.1016/j.cell.2015.05.010DOI Listing
June 2015

Dicer nuclease-promoted production of Let7a-1 microRNA is enhanced in the presence of tryptophan-containing amphiphilic peptides.

Chembiochem 2014 Jul 2;15(11):1651-9. Epub 2014 Jul 2.

Department of Chemistry and Education, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742 (Korea).

A library of Trp-containing amphiphilic peptides was synthesized and screened for the ability to bind to pre-miRNA targets. Two members of this family, peptides Ac-WKKLLKWLKKLLKLAG-NH2 (2 b) and Ac-WKKLLKWLKKLLDabLAG-NH2 (4 b) were found to have nanomolar binding affinities to pre-let7a-1. Peptides 2 b and 4 b caused an increase in the in vitro Dicer cleavage of pre-let7a-1. This observation was confirmed by a cell-based assay, the results of which show an up to 50 % increase in Dicer activity. Enhanced expression of let7a-1 promoted by the peptides results in specific reductions of target mRNAs. The results of a microarray study show that lower amount of fluctuating genes are generated in the presence of 2 b or 4 b, relative to that from exogenous delivery of let7a-1. Because peptides 2 b and 4 b promote enhanced formation of mature let7a-1 only at the endogenous miRNA level, this specifically controls genes related to let7a-1.
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http://dx.doi.org/10.1002/cbic.201402126DOI Listing
July 2014

Protein conformational dynamics dictate the binding affinity for a ligand.

Nat Commun 2014 Apr 24;5:3724. Epub 2014 Apr 24.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.

Interactions between a protein and a ligand are essential to all biological processes. Binding and dissociation are the two fundamental steps of ligand-protein interactions, and determine the binding affinity. Intrinsic conformational dynamics of proteins have been suggested to play crucial roles in ligand binding and dissociation. Here, we demonstrate how protein dynamics dictate the binding and dissociation of a ligand through a single-molecule kinetic analysis for a series of maltose-binding protein mutants that have different intrinsic conformational dynamics and dissociation constants for maltose. Our results provide direct evidence that the ligand dissociation is determined by the intrinsic opening rate of the protein.
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http://dx.doi.org/10.1038/ncomms4724DOI Listing
April 2014

Distinct Z-DNA binding mode of a PKR-like protein kinase containing a Z-DNA binding domain (PKZ).

Nucleic Acids Res 2014 May 20;42(9):5937-48. Epub 2014 Mar 20.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea

Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Zα) of PKZ from Carassius auratus (caZαPKZ). The 1.7-Å resolution crystal structure of caZαPKZ:Z-DNA revealed that caZαPKZ shares the overall fold with other Zα, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZαPKZ and its mutants revealed that caZαPKZ mediates the fastest B-to-Z transition of DNA among Zα, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZαPKZ. Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the β-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZαPKZ further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the β-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate.
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http://dx.doi.org/10.1093/nar/gku189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027156PMC
May 2014
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