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公告主旨 Joint CQSE and CASTS Seminar
公告單位 量子科學與工程研究中心
公告時間 2014/10/16 下午 02:04:07
公告內容 Joint CQSE and CASTS Seminar

TIME: Oct. 24, 14:30 ~ 15:30
TITLE: Single Molecule Observation of Direct Transfer of Escherichia coli Single-Strand Binding Protein (SSB) Between Single-Stranded DNA Molecules
SPEAKER: Prof. I-Ren Lee 李以仁教授
      Department of Chemistry, National Taiwan Normal University
      國立臺灣師範大學化學系
PLACE: Rm716, CCMS & New Physics Building, NTU
     臺大凝態大樓/物理新館 R716

Abstract:
Echerichia coli (E. coli) Single-Strand Binding (SSB) protein is essential in DNA replication and repair processes owing to its ability to bind the intermediate single stranded DNA (ssDNA), thereby preventing unwanted reannealing or degradation. It was proposed that SSB is recycled by redistributing itself along long ssDNA, for example between two adjacent Okazaki fragments. The mechanism of this process was investigated by the Lohman group using ensemble kinetics methods and a previously proposed a “direct transfer” mechanism - forming a transient intermediate composed of SSB and two ssDNA prior to the transfer was concluded. We developed a single molecule assay to examine this direct transfer in detail through real-time observation of single molecule fluorescence resonance energy transfer (smFRET) signals. The introduction of competitor ssDNA oligonucleotides to an SSB protein transiently bound to a surface immobilized ssDNA ultimately led to the dissociation of SSB from the surface immobilized DNA. The rate of SSB transfer is linearly dependent on the competitor DNA concentration, suggesting that only one ssDNA molecule is involved in the rate-determining step of the transfer process. Prior to the full unwrapping of the original DNA bound to an SSB protein, fast FRET fluctuations with ~100 ms time scale were observed with occasional sudden drops of FRET signal shifted toward the lower value, indicating the structural destabilization of the ssDNA-SSB complex induced by multiple events of competitor ssDNA binding. At the given condition of dT50-SSB complex with dT40 competing ssDNA molecules, the final dissociation event measured as a high to low FRET transition occurs rapidly. These findings are consistent with the kinetic model of short-lived and partially-unwrapped intermediate states were induced by the fast binding and unbinding of competing oligonucleotides and only fraction of the intermediate state molecules can achieve complete transfer of SSB.



**Sponsored by Center for Quantum Science and Engineering (CQSE) (量子科學與工程研究中心) and Center for Advanced Study in Theoretical Sciences (CASTS) (理論科學高等研究中心), National Taiwan University
活動時間 2014/10/24
活動地點 物理學系/凝態科學研究中心Rm716, CCMS & New Physics Building, NTU
聯絡電話 02-33669857
公告對象 教職員、學生、校友、其他
附件名稱  檔案大小 
20141024  176112 Bytes
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