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Assigned Papers: download from website:http://tfiib.med.harvard.edu/bcmp200/

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Facts (“vocabulary”)

Concepts

Quantitation

Techniques

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 itBinds??

1

Nitrogenous base

Sugar

Phosphate

1A

2. Structure of dCTP

3. Base Tautomerism

3. Chargaff rules- A=T, G=C

helical

10 layer Lines BetweenCrossPatterns(10 ResiduesPer 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

NIH(not in handout)

-2’-deoxyribose

2

Sugar “Pucker” Conformations

A DNA

B DNA

3

Pyrimidines

Purines

4

G (Keto) G (Enol) A

99.99% 0.01%

Base Tautomerization

5

BaseAdenineGuanineThymineCytosine

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

A very useful number:660

Rotation About the N-Glycosidic Bond

N3

A,B DNA Z DNA (G only)

7

A

PhosphodiesterBackbone

8

Pitch 34 Å

Rise3.4 Å

Width 20 Å

Major Groove

Minor Groove

9

10.4 bp/turn

B-DNA: A rightHanded double helixWhy?

Twist 36°

9

8.5 Å 11.7 Å

7.5 Å 5.7 Å

Major Groove

Minor Groove

10

11

Note to self:Discuss forces that affect helix

formation

C-G

T-A

NIH

A B Z

Handedness

Pitch

Base Inclination 12

dx = 0.8 Å

B DNA

Major Minor

dx = -4 Å

A DNA

Major Minor

Base Displacement Determines Groove Depth

dx = +3-4 Å

Z DNA

Major Minor

13

A B Z

Mi Ma

MaMiMi

Ma

12

A B Z

Z-DNA Phosphate Backbone is Kinked

14

15

Question: is all B-DNA structurally identical?

Implications of structural variation

Implications of flexibility

Degrees of freedom: 7 Torsion angles and sugar conformation

5’ 3’

(Rigid)

16

Structural Variation Defined by Bases

17

normal frequent never

Never (except in intercalation)

Common Common

5’

3’ 3’

3’ 3’

5’

5’

5’

Propeller Twist Maximizes Base Stacking

NIH

PropellerTwist

Buckle

18Textbook Real Life

19

Naturally Occurring Variations in Roll, Slide, Twist

Pyrimidine-Purine Steps Have Little Base StackingStep Definition: Going along one strand of DNA in 5’to 3’ directionFour Possibles: P-Y, P-P, Y-P, Y-Y

5’

3’ 3’

5’C

GA

T

19A

Purine-Pyrimidine Steps Have Extensive Base Stacking

5’

3’

3’

CA

TG

5’

19B

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.

DNA Topology*

*Johannes’ Favorite Subject(Students’ least favorite subject)

DNA Unwinding Causes Topological Problems

(Transcription)

UnwoundParentalDuplex

Over-Woundregion

20

More Topological Problems

21

Properties of Topoisomerases

22

Strand Passage Model for Topo I

UnwoundComplex

Cleavage Complex

Covalent Tyrosine-5’P

StrandPassage

Re-ligation

L=2 L=323

Topo IReactions

24

Model for Topo II Mechanism

25

Topo IIReactions

26

For a good treatment of topos, see the book:“DNA replication”Arthur Kornberg and Tania Baker