1. 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

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)

4. RecBCD Pathway

5. 實驗設計 因為 RecB,RecD 有許多共同點 weak helicase, nuclease, contain SF-1,, ATPase….  RecBCD, RecB K29Q CD, RecBCD K177Q (ATP 的利用有關 )  實驗的目的 : Highly Processive DNA Unwinding

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

7. 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, RecB K29Q CD, RecBCD K177Q and single-stranded DNA binding protein (SSB)

8. 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

9. 公式 D =the total of DNA ends E =total enzyme conc. V =unwinding rate K d =the enzyme affinity to DNA

10. 名詞介紹 Unwinding rate Unwind amplitude heparin

11. 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

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

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

15. 對各種 DNA 尾端的 接受度

16. 整理

18. 事先作 pre- bond 的動作, 把 RecB K29Q CD 先結合在 DNA 上

19. 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 RecB K29Q CD 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.

20. 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?

21. 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

22. END