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PROPERTIES OF DNA I
B. Nucleotide Conformations
PRIMARY SEQUENCE
C. Types of DNAD. SupercoilingE. Restriction nucleasesF. DNA Sequencing
A. Base Pairing
Lactim(enol)
Adenine
Guanine
Cytosine
Uracil (Thymine)
HOH
(H,CH3)OH
N
N (H,CH3)
N
N N
NHNH
NH
N
N
O
H
O
N
N N
NNH2
N
NNH2 N
N
N
N
O
OH
O N
NNH2
ON
NNH2 N
N
imino
imino
amino
keto enol
enolketo
Lactam
TAUTOMERIC FORMS
(keto)
Conformational Rules
• Nucleotides are flexible and can be twisted about their C-O-P bonds
• There are 7 torsion angles in a nucleotide• One torsion angle joins base to sugar• The deoxyribose ring is “puckered” and not
flat• Puckering influences position of PO4 on the
3’ and 5’ position of the ring
CONFORMATION OF SUGAR-PHOSPHATE
NH
H2
ON
N N
N
O
O OHH
HOCH2interaction
syn-Guanosine anti-Guanosine
HO
N
NN
N
NH2HOCH2
O
O OHH
Nucleotides in DNA have 7 torsion angles that govern orientation of nucleotide chain.
FavorableUnfavorable
Z-DNA
Torsion Angles in a Nucleotide
• 7 torsion angles• Rotation at hindered• C-O-P bonds flexible• C-C, C-O, P-O flexible
C3’-endo C2’-endo
Conformations of the Deoxyribose Ring
5’
C3’-endo is found in A-DNAC2’-endo is found in B-DNA
Note 3’ and 5’ PO4 group in C2’endogives bases an arrangement more perpendicular to the helix axis
Tilts Bases No Tilt
TYPES OF DNA
1. 3 types: A, B, and Z
2. Not in equilibrium
3. Transition depends on humidity, temperature and DNA binding proteins
B-DNA (Watson-Crick) 90% humidity
1. Two Antiparallel polynucleotide strands
2. Sugar phosphates on periphery(Minimize charge repulsion)
3. Helix approximately 20 Angstroms in diameter4. 10.5 base pairs per turn, ~36 degrees per base pair
5. Bases flat, perpendicular to axis
6. Major and minor grooves readily apparent
B-DNA
MajorMinor
A DNA:
Why is A DNA important to know?
A DNA is seen in single-stranded RNA molecules that fold back on themselves. A DNA is also seen in DNA-RNA hybrids. Low humidity causes it to form from B DNA
What distinguishes A DNA from B DNA?
A DNA is wider and flatter: 11 base-pairs per turn instead of 10.5. The helix diameter is 26 angstroms instead of 20. The major groove is narrow and subdued.
Is base-pairing the same?
But the bases join around the axis and not through the axis and are tilted 20 degrees. Yes.
Z-DNA
1. Left handed helix2. 18 Angstron diameter3. No major groove4. 12 base pairs per turn5. Repeating units is a dinucleotide dRY or dYR:
d(GC) d(CG) d(AC) d(GT)6. Formation also depends on high salt to block charge
repulsion
Z-DNA Transition region
Negative twisted DNA
CGCGCG
What forces hold a typical DNA molecule together?DNA Dialogue
ANS: Hydrogen bonds between bases either through oror around the axis and base stackingWhat is base stacking?Stacking implies vertical interactions between bases asthey sit on top of one anotherWhat sort of interactions?Mainly van der Waal forces created by hydrophobicinteractions Are the forces of interaction the same for all bases?No. Stacking interactions between G and C give rise togreater stacking energy than A to T
What does this do to the DNA?Ans: The greater the GC content of DNA the greaterthe stability, thermal stability in particular
What do you mean by thermal stability?
Two ways to view thermal stability. It could be the heatenergy required to separate or melt the strandsWhat else besides heat?
Thermal could reflect the strength of bonding of the twoDNA strands to one another though a combinationof both H-bonding and base stacking
How is thermal stability measured?Next slide
50 70 90
Hyperchromicity
A260
Tm (melting temperature)
Higher G + C
Lower G + C
Temperature oC
Melting Point of DNA
DNA-Protein Interactions
Rule: The interaction of proteins with nucleic acids is animportant biological property governing nucleic acidfunction in replication and transcription
Examples:
1. Endo and Exonucleases, kinases, ligases2. Histones3. Transcription Repressor proteins
5. Topoisomerases6. Single strand DNA binding proteins7. DNA-RNA polymerases
4. Transcription Enhancer proteins
Rule: In DNA-RNA-Protein interactions there must exist a structural harmony between the nucleic acid and the protein at points of contact
Transcription Factors1. Helix-turn-helix protein (HTH)
a. Bind to operatorsb. Typically dimericc. Repress transcription of specific genes
2. Zinc finger proteina. Eukaryoticb. Cysteine and Histidine rich
1. Typically cys2-his2
Zinc Fingers (continued)
c. Designed to recognize asymmetric base sequences
3. Leucine Zippersa. Leucine repeats every 7th residueb. Dimerizes as a coil-coil, -leucine are teethc. Basic region adjacent to zipper binds to DNAd. Differ from other transcription factors
by engaging DNA at basic end of proteine. Typical is the AP1 transcription factor
composed of c-jun and c-fos leucine zippers, related to v-jun and v-fos, known heterodimeric oncogenes
Zinc Finger
Leucine Zipper
Project intoDNA grooves
Y shape is a typicalfeature of zippers
DNA must have aninverted repeat toaccommodate bZIP
bZIP