My research focuses on DNA damage repair at the single molecule level, specifically the dynamics of DNA damage recognition proteins on DNA.
Single-molecule visualization reveals the damage search mechanism for the human
- Introduction
- Results
- XPC-RAD23B exhibits three distinct classes of motion on undamaged DNA
- The location of constrained and immobile states is correlated with consecutive AT-tracks
- XPC-RAD23B diffuses on DNA via hopping
- XPC-RAD23B can bypass protein obstacles on DNA
- XPC-RAD23B recognizes CPDs with low efficiency
- Discussion
- The motion of XPC-RAD23B along DNA is heterogenous
- XPC-RAD23B searches for local lesions via hopping
- XPC inefficiently identifies CPDs and binds them with limited stability
- Conclusion
- Experimental Methods
- Preparation of lesion-containing λ-DNA
- Purification and fluorescent labeling of proteins
- Single-molecule DNA curtain assay
- Data analysis
- Electrophoretic mobility shift assay (EMSA)
- In vitro NER assay
- Abstract
- Introduction
- Results
- TonEBP interacts with METTL3
- TonEBP is recruited to damaged DNA where it induces m6A RNA methylation
- TonEBP preferentially binds R-loops
- TonEBP depletion causes R-loop accumulation and transcription-replication conflicts
- m6A RNA methylation is specific to R-loops at DNA damage sites
- The RHD of TonEBP is important for R-loop resolution
- TonEBP-mediated m6A methylation occurs at damageinduced R-lo
- Discussion
- Experimental methods
- Tandem affinity purification (TAP) and mass spectrometry analysis
- Single-molecule DNA curtain assay
- Immunofluorescence assay for R-loop without nucleolus
- Cells and reagents
- Immunofluorescence, microscopy and image analysis
- Immunoprecipitation
- S9.6 IP
- Immunoblotting
- PLA (Proximity ligation assay
- Cell survival analysis
- The molecular combing assay
- Separation of nuclear and chromatin-bound fractions
- Buffer for in vitro assays
- Purification of TonEBP
- Electrophoretic mobility shift assay for R-loop binding of Yc1
- Quantification and statistical analysis
Using a DNA curtain containing purified TonEBP and R-loop-containing lambda DNA, I demonstrated that TonEBP identifies R-loops via both 1D diffusion and 3D collision. Furthermore, TonEBP preferentially binds the displaced ssDNA in the R-loop structure, as I confirmed using an electrophoretic mobility shift assay with various types of DNA constructs. This study shows that TonEBP recognizes R-loops on DNA and recruits R-loop elimination proteins such as METTL3-METTL14 to resolve the R-loops by RNase H1.
High‐throughput single‐molecule imaging system using nanofabricated trenches and
Introduction
To achieve higher sensitivity and throughput with biochips, the integration of single-molecule techniques has recently been attempted87. Advances in single-molecule spectroscopy have led to breakthroughs in the life sciences for several reasons. However, with single-molecule techniques, statistical validity must always be guaranteed to demonstrate that the results are compatible with those obtained using ensemble assays, and therefore many samples must be examined by repeated measurements.
A multichannel DNA curtain microfluidic system based on poly(dimethylsiloxane)89 was developed to investigate complex biological reactions. The recent replacement of nanofabrication with photolithographic tools for the fabrication of multiple sets of DNA curtains has dramatically increased the throughput90. The DNA curtain is formed on a supported lipid bilayer, which not only prevents non-specific adsorption of proteins on the surface of the slide, but also enables DNA movement under hydrodynamic force due to the fluidity of the lipid bilayer91-93.
In the early DNA curtain system, the diffusion barrier was provided by manually cut scratches, in which lipid molecules cannot transverse due to the high bending energy94. Fabrication of nanometer-sized chromium (Cr) structures as a diffusion barrier can produce a more uniform arrangement of DNA molecules (Figure 3.1A)93. Even with mild detergents, these metal barriers will be worn away after a few dozen cycles (Figure 3.1B, top).
It is also very challenging to remove sticky contaminants from the sliding surface and from Cr barriers (Figure 3.1B, middle). In addition, the metal barriers sometimes scatter the excitation beam, and significant autofluorescence can arise near the barriers due to residual electron beam resist, poly(methyl methacrylate) (PMMA), especially under illumination at 637 nm (Figure 3.1B, bottom). In most DNA curtain experiments, DNA was visualized by staining with the fluorescent intercalation agent YOYO.
A disadvantage of YOYO-1 is its rapid photobleaching under continuous laser illumination, even in the presence of anti-photobleaching agents. To address the issues with YOYO-1, we used fluorescent protein-DNA binding peptides (FP-DBP) to stain DNA. Because the fluorescent domain is separated from the DNA-binding motif, it does not induce photocleavage of DNA, allowing stable long-term observation of DNA in the curtain.
Results
- Optical performance of nanotrenches for DNA curtain
- FP–DBP for the DNA curtain formed at nanotrenches
- Mapping of protein binding using nanotrenches
In the presence of 0.1 nM FP–DBP, without any anti-photobleaching agents, λ-DNA molecules were well stained. To determine whether the mechanical property of DNA was altered by FP-DBP binding, we measured DNA persistence length versus flow rate at 0.1 nM FP-DBP (Figure 3.4D). These previous persistence length measurements were made on stained DNA after washing out free FP-DBPs.
In the presence of free FP-DBPs, increasing the flow rate stretches the DNA to greater lengths so that more FP-DBPs can bind. Consequently, the binding of FP-DBPs causes the DNA to become more rigid, increasing its persistence length. We also compared the photochemical properties of FP-DBP with those of YOYO-1 at the same concentration (0.1 nM).
We observed that FP-DBP was photobleached, indicated by a gradual decrease in its fluorescence. We measured the relative photobleaching of FP-DBP and YOYO-1 at 6.7 W/mm2 of 488 nm laser. Our results showed that FP-DBP had better photochemical properties for fluorescence imaging of DNA molecules in the DNA curtain than YOYO-1, which allowed us to observe DNA for a longer time.
The gradual disappearance of fluorescence at high pH was due to the dissociation of FP-DBP from DNA98. Similarly, FP-DBPs dissociated from DNA in the presence of high salt buffer (300 mM NaCl and 10 mM MgCl2) and returned to DNA when the buffer was exchanged to 1X TE [7.5] buffer containing FP-DBP (Figure 3.5)101 . Taken together, our data showed that FP-DBPs can be removed from DNA by high pH or salt.
We also tested EcoRI binding in the presence of FP-DBP to DNA to examine the influence of FP-DBP on protein binding (Figure 3.7). When λ‐DNA was pretreated with FP–DBP, EcoRIE111Q bound to λ‐DNA, but the number of EcoRIE111Q binding to single DNA was reduced by half (Figure 3.7AB). Our results suggested that FP-DBPs disrupt protein binding when they bind to DNA.
Discussion
Schematic of the DNA curtain system with chrome baffles. (top) A DNA curtain was formed on a Cr nanobarrier constructed in the middle of the microchannel of the flow cell. bottom). Actual image of a DNA curtain with YOYO-1-stained DNA molecules aligned on a Cr barrier. the arrow on the left side of the picture indicates the position of the barrier. top). The FP-DBP constructs consisted of a green fluorescent protein (PDB ID: 1EMA) with DNA binding motifs on both N-. The fluorophore represented by the spheres is protected by a beta-tube.
Images of a DNA curtain stained with FP-DBPs in 1X TE [7.5], without any anti-photobleaching agents. top). In flowing buffer, DNA molecules stained with FP-DBP were well aligned on the nanotrench. bottom). DNA molecules were stained with 0.1 nM FP-DBP, and DNA extension was measured at different flow rates in the presence (magenta filled circle) or absence (blue blank square) of free FP-DBP.
Plots of DNA relative elongation (
Photobleaching curves of DNA curtains stained with FP-DBP (filled square) or with YOYO-1 (open circle) at 6.7 W/mm2 of 488 nm laser. The photobleaching curves were fitted by a single exponential decay function, yielding characteristic photobleaching times for FP-DBP (τ_FP-DBP) and YOYO-1 (τ_YOYO-1) of 42.4 and 4.3 s, respectively. After injection of a pH 11.0 buffer without FP-DBP, the fluorescence of FP-. DBP rose momentarily and then fell sharply.
The arrowhead on the left side of the graph indicates the position of the nanowire. bottom) Background fluorescence kymograph under the same conditions, measured in the absence of DNA. The arrowhead to the left of the kymograph indicates the location of the nano trench.-. Total internal reflection fluorescence microscope images of DNA curtain with EcoRIE111Q bound to DNA. top) DNA molecules stained with fluorescent protein–DNA binding peptide (FP–DBP; green). middle) Puncta are EcoRIE111Q labeled Qdots (magenta). bottom).
Experimental methods
- Fabrication of nanotrenches
- Protein preparation
- Single‐molecule DNA curtain assay
Sugasawa, K.; Okamoto, T.; Shimizu, Y.; Masutani, C.; Iwai, S.; Hanaoka, F., A multistep damage recognition mechanism for global genomic nucleotide excision repair. H.; Ansari, A., Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex. E.; Broyde, S., Lesion Sensing during Initial Binding by Yeast XPC/Rad4: Towards Prediction of Resistance to Nucleotide Excision Repair.
Wood., Dual-incision assays for nucleotide excision repair using DNA with a site-specific lesion. Jo, K., Single molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins. Na Young Cheon, (2022), Investigation of R-loop recognition proteins using single molecule DNA curtain technique and electrophoretic mobility shift analysis, Methods in Molecular Biology.
강유진*, 천나영*, 차종진*, (2020) 나노 가공 트렌치 및 형광 DNA 결합 단백질을 사용한 고처리량 단일 분자 이미징 시스템. 천나영, (2019) 단일 분자 시각화는 인간 NER 단백질 XPC-RAD23B에 대한 손상 탐색 메커니즘을 보여줍니다., 핵산 연구. 교수님으로부터 과학자로서 연구에 대한 태도와 사고방식에 대해 배웠습니다.
