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Introduction

Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme Mark S. Dillingham .etal 280 37069-37077 銘傳生科 學生 — 朱祐頡 2005.11.29. Introduction. 研究動機 :double-stranded breaks RecBCD=RecB+RecC+RecD RecB  從 3’ 端開始 unwinding RecD  從 5’ 端開始 unwinding

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Introduction

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  1. Bipolar DNA Translocation Contributes to Highly ProcessiveDNA Unwinding by RecBCD EnzymeMark S. Dillingham.etal 280 37069-37077銘傳生科學生—朱祐頡2005.11.29

  2. Introduction • 研究動機:double-stranded breaks • RecBCD=RecB+RecC+RecD • RecB 從3’端開始unwinding RecD 從5’端開始unwinding 都需要ATP(RecC) • Bipolar DNA的描述 • Chi sequence (5’-GCTGGTGG)

  3. RecBCD Pathway

  4. 實驗設計 • 因為RecB,RecD 有許多共同點 weak helicase , nuclease, contain SF-1, , ATPase….  RecBCD, RecBK29QCD, RecBCDK177Q (ATP的利用有關) 實驗的目的: Highly Processive DNA Unwinding

  5. EXPERIMENTAL PROCEDURES • DNA substrates- • Proteins- • Stopped-flow dye-displacement helicase assay- • Conventional dye displacement helicase assay- • SSB-binding coupled helicase assay-

  6. DNA substrates and Proteins • PBR322 4-base 5′ overhangs 2-base 5′-overhangs blunt ends 4-base 3′overhangs No Chi sequences andλ phage DNA also • RecBCD, RecBK29QCD, RecBCDK177Q and single-stranded DNA binding protein (SSB)

  7. Stopped-flow dye-displacement helicase assay- Trisacetate ,magnesium,DDT, Hoechst33258 dye and SSB 加入2mM的ATP,反應開始進行 另外作一個相同的時驗中,加入了Heparin DNA上的螢光標記 使我們知道DNA上的 unwinding的程度

  8. 公式 D =the total of DNA endsE =total enzyme conc.V =unwinding rateK d=the enzyme affinity to DNA

  9. 名詞介紹 • Unwinding rate • Unwind amplitude • heparin

  10. 圖a為pre-bond的情況討論Heparin的影響 • 圖b上圖為pre-bond 下圖為非pre-bond

  11. Conventional dye displacement helicase assay- SSB-binding coupled helicase assay- Tris-acetate magnesium acetate, SSB, and 1 mM DTT, ATP • Tris-acetate magnesium acetate, Hoechst 33258, SSB, and DTT. λ DNA. PBR322 plasmid and λ DNA 標準化: No protein (0% unwinding) Heat-denatured DNA(100% unwinding) No protein (0% unwinding) Heat-denatured DNA(100% unwinding)

  12. 加了trap (heparin)和使用了SSB的效果 • 低濃度的Mg2+離子

  13. 對各種DNA尾端的接受度

  14. 整理

  15. 事先作pre-bond的動作,把RecBK29QCD 先結合在DNA上

  16. RESULTS • Monitoring rapid unwinding of DNA by RecBCD using a stopped-flow dye-displacement assay. • Mutation of helicase motif I in either RecB or RecD reduces the observed rate and amplitude of plasmid unwinding catalyzed by the RecBCD holoenzyme. • The RecBK29QCD enzyme will only initiate unwinding from a short 5′- ssDNA overhang structure. • Mutation of helicase motif I in either RecB or RecD severely reduces processivity of translocation by RecBCD.

  17. DISCUSSION • Rates of RecBCD-catalyzed DNA unwinding: Which DNA motor is faster? • Implications for general models of SF1 helicase activity. • Why does RecBCD enzyme employ a bipolar DNA translocation mechanism?

  18. Conclusion • 針對了RecB ,RecD這兩個motor的討論 對Highly Processive DNA Unwinding • 影響RecBCD的因子 作用在突變的protein上 • 親合性的討論及對尾端的接受度

  19. END

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