5. Does Z-DNA have any physiological meaning ?

a) Can occur adjacent to β-helical sections

)n
(G--C (G-
-
(C--G
)n (C- C)n
-G)
n

regular DNA seq = β-helix

Transition from β → Z
31
P-NMR or circular dichroism

b) Science 238 (1987) 773

Z-DNA can occur inside cells

Basic idea = If DNA is in the β-form of helix

= certain enzyme can modify it
If in Z-form = can′t be modified

6. Topology of DNA

Bent DNA (alternative β- helix)

a) Some DNA′s have short A stretches

(A)n
 (T)n
b) Best example
DNA from kinetoplast (speciallized Mit of
Trypanosomes (Tropical parasites)

Found (A) 6 (A)6 (A) 6 (A) 6

The piece of DNA had unusual electrophoretic
mobility. (run shorter than predicted)

c) The short A stretches were causing the DNA

to bend
shape change in specific region of DNA

Random seq. DNA

E.M.
Bent DNA
(A)n stretches

Distance between A stretch will enhance bending

d) Koo et al., examine distance between (A)n

stretches and effect on net bend.
Biochemistry 29(1990) 4227

( A5
N
A5
) n

Random sequence
 Examine the mobility of these constructs as
N (# Nucleotides between (A)5 runs is varied)

Bent DNA ⇒ slower mobility on electrophoresis

N 4 5 6 7

Electrophoresis

Maximal bending at N = 5

5 = N = 1/2 turn of helix

At N = 5 the (A)5 sections are on the same side

of helix

Bends at each (A)5 run are adding up

⇒ larger net bend

N = 10 minimal bend
Opposite and cancel each other
 Random A5 E.M.
N=5
A5

Minimal bend at N = 10
A5 E.M.
N = 10

Nelson et al., possible X-ray structure of bend DNA

Nature 330 (1987) 221

C. Properties of ds DNA

1. Absorb UV light Quantitate DNA

dsDNA absorbs less light than free

nucleotides (Hypochromatic effect)

Free nucleotides (40% )

ssDNA (less stacking 20-25% )
Abs
dsDNA

240 254 290 UV wavelength

2. Effect of pH
 H-bonding maximal pH4-9
Below pH4: Some bases become protonated.
Disrupts H-bonding
Above pH10 : Some bases (G,T) lose H-
Disrupt H-bonding
too low pH causes
ds DNA
pH < 2
ssDNA ( further damage
)
pH > 10
ssDNA

low pH
RNA Damage
ds
( )or
ss
Hydrolysis
pH > 9
Hydrolysis to mononucleotides

3. Centrifugation of nucleic acids

Used for separation of DNAs, RNAs and

protein: Nucleic acid complex

Two basic types:

a) Velocity centrifugation (Nonequilibrium)

most common type = sucrose graident

Perform a density (ρ) graident

 Sample

5% sucrose
centrifugation up
to 200,000 xg
20% sucrose

5% sucrose
4S
separated bands
of material 12S Hemoglobin, mRNA
16S small rRNA

20% sucrose

Separation is on the bases of MR (MW), shape,

hydrodynamic properties
Relative migration is in Svedberg (S) units.

b) Equilibrium (Isopycnic) centrifugation

Put a CsCl or Cs(SO4)2 solu in a tube and

subject to high speed centrifugation
A density gradient forms during the spin.
Uniform high speed spin ρ
CsCl
CsCl solu separated bands
CsCl form a of material
gradient in tube

Gradient forms stable where

sedimentation are different balance

ρ
CsCl

Top bottom

If CsCl solu contains DNA:

Inc
Uniform high speed spin
CsCl solu ρ
CsCl
w/ DNA CsCl forms
and DNA bands
Two DNA bands
where ρ
CsCl =
ρ
DNA

Separation not by MR

Width of band is α 1/MR

Density of DNA depends on G + C

 1.73

ρ DNA

1.69
30% G＋Ｃ 70%
In general ssDNA more dense than dsDNA and
RNA is even more dense

dsDNA
ssDNA
To resolve RNA species use Cs(SO4)2

4. Denaturation /Renaturation

a) Conversion ds → ss ⇒ denaturation or
melting

Can be brought about by :

↑ Temp Disrupt the St of H2 O
pH
Formaldehyde / urea (formamide)

Transient covalent disruption of H-bonding