1. 免疫学信息网 http://www.immuneweb.com In vivo DNA binding pattern of the Polycomb Txn Factor 1.What are the genes to which it binds? 2.How does it affect these genes? 3. What determines where it Binds??

2. 免疫学信息网 http://www.immuneweb.com 1 Nitrogenous base Sugar Phosphate

3. 免疫学信息网 http://www.immuneweb.com 1A1A 2. Structure of dCTP 3. Base Tautomerism 3.Chargaff rules - A=T, G=C helical 10 layer Lines Between Cross Patterns (10 Residues Per turn) Evidence for the Double Helix 1. Fiber Diffraction data: -Helical geometry -3.4 A º spacing (1Aº = 10 -10 m) -34 A º pitch

4. 免疫学信息网 http://www.immuneweb.com NIH (not in handout)

5. 免疫学信息网 http://www.immuneweb.com  -2’-deoxyribose 2

6. 免疫学信息网 http://www.immuneweb.com Sugar “Pucker” Conformations A DNA B DNA 3

7. 免疫学信息网 http://www.immuneweb.com Pyrimidines Purines 4

8. 免疫学信息网 http://www.immuneweb.com G (Keto) G (Enol) A 99.99% 0.01% Base Tautomerization 5

9. 免疫学信息网 http://www.immuneweb.com Base Adenine Guanine Thymine Cytosine Nucleoside (Deoxy)adenosine (Deoxy)guanosine (Deoxy)thymidine (Deoxy)cytidine Nucleotide (d)A (mono, di-, tri) phosphate (d)G (mono, di-, tri) phosphate (d)T (mono, di-, tri) phosphate (d)C (mono, di-, tri) phosphate 6 1’ 9

10. 免疫学信息网 http://www.immuneweb.com A very useful number: 660

11. 免疫学信息网 http://www.immuneweb.com Rotation About the N-Glycosidic Bond N3 A,B DNAZ DNA (G only) 7 A

12. 免疫学信息网 http://www.immuneweb.com Phosphodiester Backbone 8

13. 免疫学信息网 http://www.immuneweb.com Pitch 34 Å Rise 3.4 Å Width 20 Å Major Groove Minor Groove 9 10.4 bp/turn B-DNA: A right Handed double helix Why?

14. 免疫学信息网 http://www.immuneweb.com Twist 36° 9

15. 免疫学信息网 http://www.immuneweb.com 8.5 Å11.7 Å 7.5 Å5.7 Å Major Groove Minor Groove 10

16. 免疫学信息网 http://www.immuneweb.com 11

17. 免疫学信息网 http://www.immuneweb.com Note to self: Discuss forces that affect helix formation

18. 免疫学信息网 http://www.immuneweb.com C-G T-A NIH

19. 免疫学信息网 http://www.immuneweb.com ABZ Handedness Pitch Base Inclination 12

20. 免疫学信息网 http://www.immuneweb.com dx = 0.8 Å B DNA MajorMinor dx = -4 Å A DNA MajorMinor Base Displacement Determines Groove Depth dx = +3-4 Å Z DNA MajorMinor 13

21. 免疫学信息网 http://www.immuneweb.com ABZ Mi Ma Mi Ma 12

22. 免疫学信息网 http://www.immuneweb.com ABZ Z-DNA Phosphate Backbone is Kinked 14

23. 免疫学信息网 http://www.immuneweb.com 15

24. 免疫学信息网 http://www.immuneweb.com Question: is all B-DNA structurally identical? Implications of structural variation Implications of flexibility

25. 免疫学信息网 http://www.immuneweb.com Degrees of freedom: 7 Torsion angles and sugar conformation 5’ 3’ (Rigid) 16

26. 免疫学信息网 http://www.immuneweb.com Structural Variation Defined by Bases 17 normal frequentnever Never (except in intercalation) Common

27. 免疫学信息网 http://www.immuneweb.com 5’ 3’ 5’ Propeller Twist Maximizes Base Stacking NIH

28. 免疫学信息网 http://www.immuneweb.com Propeller Twist Buckle 18 TextbookReal Life

29. 免疫学信息网 http://www.immuneweb.com 19 Naturally Occurring Variations in Roll, Slide, Twist

30. 免疫学信息网 http://www.immuneweb.com Pyrimidine-Purine Steps Have Little Base Stacking Step Definition: Going along one strand of DNA in 5’to 3’ direction Four Possibles: P-Y, P-P, Y-P, Y-Y 5’ 3’ 5’ C GA T 19A

31. 免疫学信息网 http://www.immuneweb.com Purine-Pyrimidine Steps Have Extensive Base Stacking 5’ 3’ C A T G 5’ 19B

32. 免疫学信息网 http://www.immuneweb.com For further reading on effects of sequence on structure, “Understanding DNA-The Molecule and How it Works” By Calladine and Drew Major Conclusion: DNA structure can depend on sequence In predictable, yet complicated ways. Therefore, DNA binding proteins can recognize structure, And they can be designed to bind to highly flexible DNA.

33. 免疫学信息网 http://www.immuneweb.com DNA Topology* *Johannes’ Favorite Subject (Students’ least favorite subject)

34. 免疫学信息网 http://www.immuneweb.com DNA Unwinding Causes Topological Problems (Transcription) Unwound Parental Duplex Over- Wound region 20

35. 免疫学信息网 http://www.immuneweb.com More Topological Problems 21

36. 免疫学信息网 http://www.immuneweb.com Properties of Topoisomerases 22

37. 免疫学信息网 http://www.immuneweb.com Strand Passage Model for Topo I Unwound Complex Cleavage Complex Covalent Tyrosine-5’P Strand Passage Re- ligation L=2 L=3 23

38. 免疫学信息网 http://www.immuneweb.com Topo I Reactions 24

39. 免疫学信息网 http://www.immuneweb.com Model for Topo II Mechanism 25

40. 免疫学信息网 http://www.immuneweb.com Topo II Reactions 26

41. 免疫学信息网 http://www.immuneweb.com For a good treatment of topos, see the book: “DNA replication” Arthur Kornberg and Tania Baker