Study of molecular mechanism of nucleases
Dynamic coordination of two-metal-ions orchestrates lambda-exonuclease catalysis
- Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity. we use λ-exonuclease as a model enzyme with two Mg2+ binding sites and probe activity at various concentrations of magnesium by single molecule-FRET
- Our study highlights the importance of metal ion-coordination dynamics in correlation with the enzymatic reaction-steps, and offers insights into the origin of dynamic heterogeneity in enzymatic catalysis
Proofreading mechanism of phi29 DNA polymerase based on DNA stability
- Phi29 DNAP adopts two binding modes, representing for its polymerase and exonuclease activities, and the switching between these two modes takes places intramolecular. The intramolecular switching behavior can maximize the efficiency of mismatch recognition and removal.
- A wide-range of mismatch recognition reduces the probability of mismatch “stalling”, resulting the high fidelity of enzyme.
Unwinding mechanism of SARS Coronavirus helicase nsP13
- SARS was epidemic in 2003 worldwide. SARS-CoV helicase (nsP13) plays critical roles in viral replication, and has been proposed to be a potential candidate for anti-SARS therapy. Thus, the characterization of enzymatic activity might be critical for drug development
- We use single molecule fluorescence resonance energy transfer to characterize the behavior of nsP13 helicase
Binding mode and degradation activity of Ribonuclease H on the RNA/DNA hybrid
- Ribonuclease H (RNase H), which is known as a nuclease enzyme is found in all organisms. The RNase H malfunctioning induces the autoimmune disease such as Aicardi-Goutieres syndrome (AGS). This suggests RNase H is necessary component for fundamental cellular process
- The main function of RNase H is to remove RNA primers in Okazaki fragments during replication. However, how RNase H recognizes small RNA primer fragments in long lagging strands remains uncertain.
- Single molecule FRET was used to observe binding mode of RNase H with various substrates.
Development of biosensor for early diagnosis of cancers and study in biomarkers of cancers
Molecular mechanism of AP site recognition by exonuclease III during Base Excision Repair
- Ape1 and Exo3 are a kinds of AP endonuclease which is one of the most important enzyme to recognize AP site during BER mechanism. Moreover, Ape1 (Exo3) is related with cancer very well and over-expressed in cancer cells. Despite many studies of AP endonuclease, how they recognize AP sites is still poorly understood.
- We focused on exonuclease III which has one of the AP endonuclease activity and already well-studied how those nucleases are associated each other and can recognize only AP site specifically.
The new designed biosensor for early diagnosis of cancer
- To develop the biosensor by measuring the Ape1 level in cells, we designed the short oligo dsDNA conjugated biotins and labeled by Cy3 to see the change in high level of Ape1. We also interact the oligo dsDNA with NeutrAvidin to block the end of dsDNA parts and to react the enzyme only in AP site. Designed oligo dsDNA also can occur the FE(Fluorescence enhancement) effect when dsDNA change to the ssDNA by the activity of Ape1 and 3’to 5’ exonuclease on the AP site .
- We quantified specificity and sensitivity of the Ape1 in designed biosensor by FE effect. Finally, we expect that the biosensor can be a magnificent sensor which can diagnose cancer cells in normal cells well
The development of platform to detect G-Quardruplex(GQ) mutation
- GQ is a DNA or RNA secondary structure which is found frequently in telomeres and gene promoter regions. Because GQs act as biological regulators, the mutations of GQ can result in cancers or diseases. Oxidative DNA damage is the most common mutation at GQ. 8-oxoguanine (8oxoG) is a most common oxidative damage in GQ structures which affects the stability of GQ structures.
- Using this property of stability, We are developing the simpler and easier platform to detect mutations such as 8oxoG. We have established a fluorescence analysis method using GQ interacting compounds called Crystal Violet (CV) and N-Methylmesoporphyrin IX(NMM). In the future, we will develop this platform by using enzyme and try to different conformation GQ mutation.
Spatiotemporal analysis of signaling pathways by superresolution imaging
Elucidating the mechanism of EMT Homeostasis by TGF-β superfamily signaling
- Currently, over 40 ligands have been identified in TGFβ superfamily. These ligands can initiate signaling by binding to different set of type1/2 receptor hetero-tetramers, and phosphorylating SMADs. Interestingly, these diverse and complicated binding pattern of ligand and receptors converges on only two SMAD pathways, which are SMAD2/3 and SMAD1/5/8. In this research we want to understand how the diverse response of various TGFβ superfamily ligand-receptor binding can be drawn by these simple two pathway system.
- Using DNA-PAINT super-resolution microscopy, we can image subcellular structures upto vesicular resolution, which enables us to track down the receptor internalization process, and to identify each type1/2 receptor pairs. Furthermore, DNA-PAINT technique can use large number of markers in one sample, allowing identification of multiple receptor in single sample.