DNA Double-Strand Breaks Trigger Genome-Wide Sister-Chromatid Cohesion Through Eco1(Ctf7)

1. ------《Science》 13 July 2007 DNA Double-Strand Breaks Trigger Genome-Wide Sister-Chromatid Cohesion Through Eco1(Ctf7) 金漫 王晓艳 沙龙泽 2007-11 -20

2. what factor acts here to bind sister-chromatid together? Cohesin

3. It is a model of cohesin The difference between two key words in this paper: Cohesin Cohesion(粘连过程): is a process(过程)

4. Today we know The cohesin have two functions: ① Faithful chromosome segregation (染色体间的分离) ② repair of DNA double-strand breaks(DSBs) ① is induced by DNA replication ② is induced by DSB

5. Function 1: Faithful chromosome segregation Centromere cohesin Arm cohesin S-Cohesin The word “S” means S-phase of the cell cycle

6. Function 2: repair of DNA double-strand breaks(DSBs) This kind of cohesin is induced by DSBs The DSB-cohesin is used here to repair the DNA damage DSB-cohesin

7. Remember : Both S-cohesin and DSB-cohesin are the same kind Induction pathway is different

8. Questions • 1 Whether DNA replication is required for DSB-induced cohesion? (Relationship) • 2 By the induce of DSBs. cohesion occur only around broken chromosomes and not on unbroken chromosome? • 3 Eco1(cohesion establishment factor) ,How to mediate the process of cohesion?

9. Experiment 1: The cohesin which is induced by DNA replication has a thermo-sensitive(温度敏感型突变) subunit mcd1-1 Wild-type

10. Actually the result is there is only one dot • So it can be identified that the DSBs is a another pathway can induce cohesion

12. Up to 60% Loss Go back to 20% Why not 100% cohesion?

13. Conclusion of Experiment 1 • 1 Cohesion can be generate outside of S-phase • 2 Both DSBs and wild-type cohesion(S-cohesin) are necessary, but neither is sufficient • 3 As few as two DSBs are sufficient to induce cohesin-dependent cohesion in G2/M

14. Questions • 1 Whether DNA replication is required for DSB-induced cohesion? (Relationship) • 2 By the inducement of DSBs,whether cohesion can occur only around broken chromosomes and not on unbroken chromosomes? • 3 What is the key factor for the cohesion establishment?

15. Experiment 2 • We move the cohesion reporter (LacI-GFP) to chr.Ⅰwhile keeping the HO-cs on chrⅢ. We found that though there is no DSB on chr.Ⅰ, cohesion is still established by the induction of DSBs on chr. Ⅲ

16. Conclusion of experiment 2 • Cohesion can occur both around broken chromosomes and on unbroken chromosomes genome-wide cohesion • This generation of genome-wide cohesion cannot occur by a replication-dependent mechanism • DSBs can trigger genome-wide sister-chromatid cohesion

17. Background of Experiment 3 Nucleosome (DNA+histone) • DNA damage response pathway • Key protein: MRE11 MEC1 H2A TEL1

18. Experiment 3 • We examined damage-induced cohesion in cells mutated for MRE11, MEC1 , H2AX and TEL1.

19. Results of experiment 3 • 1.In mer11△ or mec1△ cells, cohesion fails to form on chr.ⅩⅥ in response to DSBs on chr.Ⅲ.(△ means absent) 2.In Tell △ and H2A mutated cells, the loss rate remains the same as thee wild-type cell.

20. Conclusion of experiment 3 • The chromatin-bound cohesion complex is converted to a cohesive state by the DNA damage response pathway(mer11and mec1 are necessary), presumably through a trans-acting factor.

21. Questions • 1 Whether DNA replication is required for DSB-induced cohesion? (Relationship) • 2 By the inducement of DSBs,whether cohesion can occur only around broken chromosomes and not on unbroken chromosomes? • 3 What is key factor for the cohesion establishment? (the trans-acting factor)

22. One candidate • Eco1 An essential factor for cohesion establishment in S phase. • Now we will do another experiment to identify whether Eco1 is also the key factor for DSB-induced cohesion establishment in G2/M.

23. Experiment 4 • S-cohesion inactive • without either DSB or Eco1,loss rate is high • with both DSB and Eco1,loss rate is lower

24. Experiment 4 • S-cohesion inactive Eco1 ack- acetyltransferase (乙酰基转移酶) • - or + DSB, loss rates are both high,and almost the same

25. Conclusion of experiment 4 So Eco1 is the key factor for DSB-induced cohesion and the acetyltransferase (乙酰基转移酶) is the key domain.

26. Ecol has at least two distinct biological functions: • Converts the chromatin-bound cohesion complex to a cohesive state • The acetyltransferase function activates directly or indirectly its cohesive function during G2/M.

27. Summary 1 • Cohesion is generated by an Eco1-dependent but replication-independent process in response to DSBs in G2/M

28. Summary 2 • Eco1 has two functions: • a cohesive activity • a conserved acetyltransferase(乙酰基转移酶) activity, which triggers the generation of cohesion in response to the DSB and the DNA damage checkpoint.

29. Summary 3 • The DSB-induced cohesion is not limited to broken chromosomes but occurs also on unbroken chromosomes, suggesting that the DNA damage checkpoint through Eco1 provides genome-wide protection of chromosome integrity.

30. Conclusion With DSB,the DNA damage checkpoint initiates a signal that induces wild-type cohesin loading around the DSB and the Eco1 is reactive. In response to DSBs,the Eco1 acetyltransferase activity is increased through the DNA damage checkpoint. Replication fork Cohesion is generated by the replication-driven process and Eco1 ;Eco1 can directly converts the cohesin to its cohesive state,making the sister-chromatides get together

31. Furthermore • Pay more attention to: • Where is the acetyltransferase domain? • What are the inhibitors that limit the activity of Eco1 in G2/M on unbroken chromosomes? • How does Eco1 work in S-phase? • Further studies of genome-wide cohesion may reveal additional functions such as the preventions of rearrangements through ectopic recombination Remainunclear now

32. Thank you!