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Announcements Assigned Papers: download from website: http://tfiib.med.harvard.edu/bcmp200/ Sign-up sheets


Facts (“vocabulary”) Concepts Techniques Quantitation


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??


Nitrogenous base Sugar Phosphate

1


Evidence for the Double Helix 1. Fiber Diffraction data: -Helical geometry -3.4 A ยบ spacing (1Aยบ = 10-10 m) -34 A ยบ pitch 2. Structure of dCTP 3. Base Tautomerism

helical 10 layer Lines Between Cross Patterns (10 Residues Per turn)

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

1A


NIH (not in handout)


β-2’-deoxyribose

2


Sugar “Pucker” Conformations A DNA

B DNA

3


Pyrimidines

Purines

4


Base Tautomerization

G (Keto)

G (Enol)

99.99%

0.01%

A

5


9 1’

Base Adenine Guanine Thymin e 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


A very useful number: 660


Rotation About the N-Glycosidic Bond

N3

A

A,B DNA

Z DNA (G only)

7


8

Phosphodiester Backbone


Rise 3.4 Å

Minor Groove

B-DNA: A right Handed double helix Why?

Major Groove

Pitch 34 Å 10.4 bp/turn

Width 20 Å

9


Twist 36째

9


8.5 Å

7.5 Å

11.7 Å

Major Groove

5.7 Å

Minor Groove

10


11


Note to self: Discuss forces that affect helix formation


C-G

T-A

NIH


A

B

Z

Pitch

Base Inclination

Handedness

12


Base Displacement Determines Groove Depth

A DNA

Major

Minor

dx = -4 Å

B DNA

Major

Minor

dx = 0.8 Å

Z DNA

Major

Minor

dx = +3-4 Å

13


A

B

Mi

Z

Ma

Ma Mi

Mi

Ma

12


Z-DNA Phosphate Backbone is Kinked

A

B

Z

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 (Rigid)

5’

3’

16


Structural Variation Defined by Bases normal

frequent

Never (except in intercalation)

Common

never

Common

17


Propeller Twist Maximizes Base Stacking

5’ 3’

3’ 5’

5’ 3’

3’ 5’

NIH


Buckle

Propeller Twist

Textbook

Real Life

18


Naturally Occurring Variations in Roll, Slide, Twist

19


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

3’

5’ C

T

A

G

5’ 3’

19A


Purine-Pyrimidine Steps Have Extensive Base Stacking

3’

5’ A C

3’ G

T

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

Unwound Parental Duplex

(Transcription)

OverWound region

20


More Topological Problems

21


Properties of Topoisomerases

22


Strand Passage Model for Topo I Covalent Tyrosine-5’P

Unwound Complex

Cleavage Complex

Strand Passage

L=2

Religation

L=3

23


Topo I Reactions

24


Model for Topo II Mechanism

25


Topo II Reactions

26


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


PowerPoint Presentation