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Bipolar DNA Translocation Mechanism Enhances Processive Unwinding by RecBCD Enzyme

This study investigates how bipolar DNA translocation contributes to the highly processive unwinding of DNA by the RecBCD enzyme. We explore the roles of RecB and RecD in unwinding mechanisms, focusing on their ATP dependence and interaction with DNA substrates. Experimental procedures include various helicase assays to quantify unwinding rates and amplitudes. Results demonstrate that mutations in helicase motifs decrease unwinding efficiency, shedding light on the kinetics of RecBCD's DNA motor activity and its implications for understanding SF1 helicase functions.

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Bipolar DNA Translocation Mechanism Enhances Processive Unwinding by RecBCD Enzyme

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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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