LECT29 DNA2

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Transcript LECT29 DNA2

PROPERTIES OF DNA I
PRIMARY SEQUENCE
A. Base Pairing
B. Nucleotide Conformations
C. Types of DNA
D. Supercoiling
E. Restriction nucleases
F. DNA Sequencing
NH2
NH imino
N
N
Adenine
N
N
O
Lactam
(keto)
Guanine
OH enol
N
N
N
N
N
N
NH2
NH2 amino
O
N
N
keto
HN
NH2
N
HN
N
N
NH imino
Lactim
(enol)
HN
O
N
N
Cytosine
O
(H,CH3)
HN
O
Uracil (Thymine)
keto
N
OH enol
(H,CH3)
N
O
N
TAUTOMERIC FORMS
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
Nucleotides in DNA have 7 torsion angles that
govern orientation of nucleotide chain.
O
O
HN
H2N
interaction
N
N
N
N
N
HOCH2
O
HOCH2
O
N
NH2
Favorable
Unfavorable
HO OH
Z-DNA
syn-Guanosine
NH
HO OH
anti-Guanosine
Torsion Angles in a Nucleotide
•
•
•
•
7 torsion angles
Rotation at  hindered
C-O-P bonds flexible
C-C, C-O, P-O flexible
Conformations of the Deoxyribose Ring
Tilts Bases
No Tilt
5’
C3’-endo
C2’-endo
C3’-endo is found in A-DNA
C2’-endo is found in B-DNA
Note 3’ and 5’ PO4 group in C2’endogives bases an
arrangement more perpendicular to the helix axis
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
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 diameter
4. 10.5 base pairs per turn, ~36 degrees per base pair
5. Bases flat, perpendicular to axis
6. Major and minor grooves readily apparent
Major
Minor
A 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?
Yes. But the bases join around the axis and not through
the axis and are tilted 20 degrees.
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
Z-DNA
1. Left handed helix
2. 18 Angstron diameter
3. No major groove
4. 12 base pairs per turn
5. 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
CGCGCG
Negative twisted DNA
DNA Dialogue
What forces hold a typical DNA molecule together?
ANS: Hydrogen bonds between bases either through or
or around the axis and base stacking
What is base stacking?
Stacking implies vertical interactions between bases as
they sit on top of one another
What sort of interactions?
Mainly van der Waal forces created by hydrophobic
interactions
Are the forces of interaction the same for all bases?
No. Stacking interactions between G and C give rise to
greater stacking energy than A to T
What does this do to the DNA?
Ans: The greater the GC content of DNA the greater
the stability, thermal stability in particular
What do you mean by thermal stability?
Two ways to view thermal stability. It could be the heat
energy required to separate or melt the strands
What else besides heat?
Thermal could reflect the strength of bonding of the two
DNA strands to one another though a combination
of both H-bonding and base stacking
How is thermal stability measured?
Next slide
Melting Point of DNA
Lower G + C
Higher G + C
A260
Temperature oC
50
Hyperchromicity
70
90
Tm (melting temperature)
DNA-Protein Interactions
Rule: The interaction of proteins with nucleic acids is an
important biological property governing nucleic acid
function in replication and transcription
Examples:
1.
2.
3.
4.
Endo and Exonucleases, kinases, ligases
Histones
Transcription Repressor proteins
Transcription Enhancer proteins
5. Topoisomerases
6. Single strand DNA binding proteins
7. DNA-RNA polymerases
Rule: In DNA-RNA-Protein interactions there must
exist a structural harmony between the nucleic acid and
the protein at points of contact
Transcription Factors
1. Helix-turn-helix protein (HTH)
a. Bind to operators
b. Typically dimeric
c. Repress transcription of specific genes
2. Zinc finger protein
a. Eukaryotic
b. Cysteine and Histidine rich
1. Typically cys2-his2
Zinc Fingers (continued)
c. Designed to recognize asymmetric
base sequences
3. Leucine Zippers
a.
b.
c.
d.
Leucine repeats every 7th residue
Dimerizes as a coil-coil, -leucine are teeth
Basic region adjacent to zipper binds to DNA
Differ from other transcription factors
by engaging DNA at basic end of protein
e. 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
Project into
DNA grooves
Y shape is a typical
feature of zippers
bZIP
Zinc Finger
DNA must have an
inverted repeat to
accommodate bZIP
Leucine Zipper