In vivo DNA binding pattern of the Polycomb Txn Factor What are the genes to which it binds? How does it affect these genes? 3. What determines where it Binds??

Nitrogenous base Sugar Phosphate 1

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

NIH (not in handout)

-2’-deoxyribose 2

Sugar “Pucker” Conformations A DNA B DNA 3

Pyrimidines Purines 4

Base Tautomerization G (Keto) G (Enol) A 99.99% 0.01% 5

6 9 1’ 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 生物秀 www.bbioo.com

A very useful number: 660 生物秀 www.bbioo.com

7 Rotation About the N-Glycosidic Bond A A,B DNA Z DNA (G only) N3 生物秀 www.bbioo.com

8 Phosphodiester Backbone 生物秀 www.bbioo.com

9 B-DNA: A right Handed double helix Why? Minor Groove Major Groove Rise 3.4 Å Minor Groove B-DNA: A right Handed double helix Why? Major Groove Pitch 34 Å 10.4 bp/turn 9 Width 20 Å 生物秀 www.bbioo.com

Twist 36° 9 生物秀 www.bbioo.com

10 Major Groove Minor Groove 8.5 Å 11.7 Å 7.5 Å 5.7 Å 生物秀 www.bbioo.com

11 生物秀 www.bbioo.com

Note to self: Discuss forces that affect helix formation 生物秀 www.bbioo.com

C-G T-A NIH 生物秀 www.bbioo.com

A B Z Pitch Base Inclination Handedness 12 生物秀 www.bbioo.com

13 Base Displacement Determines Groove Depth A DNA B DNA Z DNA dx = -4 Å A DNA Major Minor dx = 0.8 Å B DNA Major Minor Z DNA Major Minor dx = +3-4 Å 13 生物秀 www.bbioo.com

A B Z Mi Ma Ma Mi Mi Ma 12 生物秀 www.bbioo.com

Z-DNA Phosphate Backbone is Kinked 14 生物秀 www.bbioo.com

15 生物秀 www.bbioo.com

Question: is all B-DNA structurally identical? Implications of structural variation Implications of flexibility 生物秀 www.bbioo.com

16 5’ 3’ Degrees of freedom: 7 Torsion angles and sugar conformation (Rigid) 5’ 3’ 16 生物秀 www.bbioo.com

(except in intercalation) Structural Variation Defined by Bases normal frequent never Never (except in intercalation) Common Common 17 生物秀 www.bbioo.com

NIH Propeller Twist Maximizes Base Stacking 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 生物秀 www.bbioo.com

Buckle Propeller Twist 18 Textbook Real Life 生物秀 www.bbioo.com

19 Naturally Occurring Variations in Roll, Slide, Twist 生物秀 www.bbioo.com

19A Pyrimidine-Purine Steps Have Little Base Stacking 3’ 5’ C G A T 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’ C G A T 19A 生物秀 www.bbioo.com

19B Purine-Pyrimidine Steps Have Extensive Base Stacking 3’ 5’ A G C T 生物秀 www.bbioo.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. 生物秀 www.bbioo.com

DNA Topology* *Johannes’ Favorite Subject (Students’ least favorite subject) 生物秀 www.bbioo.com

20 DNA Unwinding Causes Topological Problems Unwound Parental Duplex (Transcription) Over- Wound region 20 生物秀 www.bbioo.com

More Topological Problems 21 生物秀 www.bbioo.com

Properties of Topoisomerases 22 生物秀 www.bbioo.com

23 Strand Passage Model for Topo I L=2 L=3 Covalent Tyrosine-5’P Re- ligation Unwound Complex Cleavage Complex L=2 L=3 23 生物秀 www.bbioo.com

Topo I Reactions 24 生物秀 www.bbioo.com

Model for Topo II Mechanism 25 生物秀 www.bbioo.com

Topo II Reactions 26 生物秀 www.bbioo.com

For a good treatment of topos, see the book: “DNA replication” Arthur Kornberg and Tania Baker 生物秀 www.bbioo.com