Announcements
Assigned Papers,download from website:
http://tfiib.med.harvard.edu/bcmp200/
Sign-up sheets
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 it
Binds
1
Nitrogenous base
Sugar
Phosphate
1A
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
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
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
A very useful number:
660
Rotation About the N-Glycosidic Bond
N3
A,B DNA Z DNA (G only)
7
A
Phosphodiester
Backbone
8
Pitch
34 ?
Rise
3.4 ?
Width 20 ?
Major
Groove
Minor
Groove
9
10.4 bp/turn
B-DNA,A right
Handed double helix
Why?
Twist 36°
9
8.5 ? 11.7 ?
7.5 ? 5.7 ?
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 ?
B DNA
Major Minor
dx = -4 ?
A DNA
Major Minor
Base Displacement Determines Groove Depth
dx = +3-4 ?
Z DNA
Major Minor
13
A B Z
Mi Ma
MaMi
Mi
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
Propeller
Twist
Buckle
18Textbook Real Life
19
Naturally Occurring Variations in Roll,Slide,Twist
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’ 3’
5’C
GA
T
19A
Purine-Pyrimidine Steps Have Extensive Base Stacking
5’
3’
3’
C
A
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)
Unwound
Parental
Duplex
Over-
Wound
region
20
More Topological Problems
21
Properties of Topoisomerases
22
Strand Passage Model for Topo I
Unwound
Complex
Cleavage
Complex
Covalent Tyrosine-5’P
Strand
Passage
Re-
ligation
L=2 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