2P119 Single-molecule visualization of RuvB oligomer for characterizing a AAA^+ class hexameric ATPase with zero-mode waveguides(04. Nucleic acid binding proteins,Poster)
RNA recognition motif (RRM), composed of a βαββαβ fold, is the most abundant RNA-binding unit in higher vertebrates, which describes about 1 % of human genome. Each of RRMs recognizes its own target nucleotide sequence with distinct specificity, and interestingly, many of RRM-containing proteins are equipped with multiple copies of RRMs. Despite this, functional significance of such "RRM multiplicity" remains obscure. Here we have noted TDP-43 as a model protein containing two RRMs in tandem
... M1 and RRM2) and developed a new assay system to evaluate the binding of each RRM with various nucleotide sequences. RRM1 but not RRM2 was found to bind nucleotides, and structural analysis describing such distinct binding capabilities of RRM1/2 is now underway. 2P116 部位特異的 RNA 切断酵素 Ire1p によって認識される HAC1 mRNA の NMR 解析 NMR analysis of HAC1 mRNA recognized by the site-specific endonuclease Ire1p The consensus sequence of HAC1, 'CNGNNGN', is cleaved at the single specific site by yeast endonuclease Ire1p. Interestingly, HAC1 mRNA has ten CNGNNGN sequences, but Ire1p cleaves two intron-exon junctions of HAC1 mRNA correctly. Here, we attempted to uncover the molecular mechanism how the specific processing sites of this HAC1 mRNA are selected. To probe the roles of the conserved sequences of HAC1 mRNA, we recorded the NMR spectra of HAC1 mRNA with the conserved residues 15 N-labeled. Based on our NMR data, we found that the sequence around this consensus sequence took a stem-loop structure where the consensus sequence is included in the loop region. This finding helps to explain the specific interaction between Ire1p and HAC1 mRNA. Single-molecule direct visualization of the mammalian nucleotide excision repair protein XPC Although XPC protein complexed with the RAD23B protein is known to be responsible for damage recognition in mammalian nucleotide excision repair, it is unclear how the protein complex discriminates base lesions from a vast excess of normal bases. To address this issue, we labeled XPC with a Qdot and developed a PEG-based single-molecule imaging platform. We found that the XPC protein complex preferably bound to DNA lesions on λDNA created by UV-irradiation. We also found that the protein complex performed one-dimensional free diffusion on undamaged λDNA. The obtained diffusion coefficient indicates that the protein complex moves on DNA by hopping as well as scanning, suggesting that the protein complex uses these modes for efficient search of DNA lesions. 2P118 ナノ開口基板を用いたヘミメチル CpG 認識に関与する SRA- DNA 複合体の機能解析 DNA modification such as CpG methylation regulates chromatin structures and eukaryotic gene expression. Many eukaryotic genes contain highly GC-rich sequences and methylation of cytosine at CpG dinucleotides is essential for silencing of parasitic DNA, genomic imprinting and embryogenesis. DNA methyltransferase 1 (Dnmt1) is the enzyme to methylate hemi-methylated CpG regions. Uhrf1 is methylated CpG binding protein and interacts with Dnmt1, followed by recruitment of Dnmt1 to hemi-methylated CpG regions. SRA domain of Uhrf1 is responsible for hemi-methylated CpG binding activity. We characterize the process of hemi-methylated CpG recognition by SRA domain using Single-Molecule technique, and in this meeting, we will show our present data. E. coli RuvB, which is classified AAA + proteins family, promotes branch migration of Holliday junction DNA together with RuvA in homologous recombination. However, despite of the importance of RuvB oligomer ring formation for its activity, the formation mechanism is poorly understood. To clarify the reaction mechanism of Holliday junction DNA branch migration by RuvA-RuvB, we prepared Cy5-RuvB and zero-mode waveguides (ZMWs) consisting of a nano-hole array. We observed interaction between Cy5-RuvB and Holliday junction, and determined the number of RuvBs under various nucleotide conditions in ZMWs. Our data demonstrated that RuvBs form higher oligomers in the presence of ATPγS and ADP, compared with those in the presence of ATPγS or ADP.