Components of DNA

Hydrogen bonding

Molecular Biology of Gene Watson et al ©

Preferred Tautomer

enol keto

Molecular Biology of Gene Watson et al © amino imino

 Structure of DNA

On

in

The space-filling model

of the double helix

Molecular Biology of Gene Watson et al ©

 Non-Watson-Crick Base Pairing

A T T

A T
A
Hoogsteen base pairing
 EE

E loop is observedin the

Rn
Sarin ricin loop 57249 23s
which involvedin Ev Tu EF a binding

k turns pseudoknots
Bates

N
 Major and Minor Grove in DNA
N

ADAM =A:T AADH = G:C

MADA =T:A HDAA= C:G
A=H-bond Acceptor
D=H bond Donor
M=Methyl group
Molecular Biology of Gene Watson et al © H=non polar hydrogen
 Multiple Conformations of DNA Double Helix

Propeller twist
NaCl >2M;
MgCl2 >0.7 M

Molecular Biology of Gene Watson et al ©

 Comparison of different forms of DNA

A DNA B DNA
RIVA
Molecular Biology of Gene Watson et al ©
 r 5th
The axis is the center the base
 1 I

it of ÉÉt
HEAD Hyped D
o o
1
OF Mon 20
CBased
plot

Kyrie K 517
o Kyrie
oh
0
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On

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9
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0 40 0
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o

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the i
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Identify Right handed and Left handed DNA

1 2 3
 Properties of DNA
UV light absorbance properties : Hypochromic and hyperchromic effects
A260 is proportional to concentration, with value of 0.02 units per microL
A260 Double-stranded DNA= 1.00
E extinctincettient
song mi A260 Single-stranded DNA = 1.37
A260 Free base = 1.60
SsDNA 0 027
DNA melting curve?
Hydrogen bonding and base stacking stabilize the DNA helix structure and
work in cooperative manner
Substances that destabilize the DNA helix

FINE Urea, Formamide, alcohols,

Evidences for H-bonds and Base stacking

Cooperatively of Base stacking

helix-destabilizing melting proteins (Gene 32:T4)

SSB-Single strand binding proteins (DNA replication)

Molecular Biology David Freifelder ©

 DNA melting cure

Japon

t.is 1

Nature ofsolvent

ph of the solan

IT
ÉEÉÉÉÉ
Denaturation of DNA first order kinetics
dsDNA Second order kinetic
Renaturation of
AT E A LAY
compliment
Iole ate my stand
one tholeades
 Effect of Ionic strength on DNA melting
Na+ form "clouds" of charge around the negatively charged phosphates
and effectively shield the phosphates from one another.

Molecular Biology David Freifelder ©

DNA Renaturation
q Salt concentration must be high to overcome electrostatic repulsion
between the phosphates (0.15 to 0.50 M) NaCl.
qThe optimal temperature for renaturation is 20-25° below the value of Tm
1A 1B 2 1C 1A 1B 2 1C
ATGA…..ATGA…..CCCC…..ATGA….. ATGA…..ATGA…..CCCC…..ATGA
TACT…...TACT…..GGGG….TACT….. TACT…...TACT…..GGGG….TACT…..
1A’ 1B’ 2’ 1C’ 1A’ 1B’ 2’ 1C’
Random Collision
At elevated Temp there is sampling of complementary base pairing/stacking in neighbor

Renaturation follows law of mass action (ie: concentration dependent)

 DNA Hybridization
Application in Southern & Northern Blot

Efficacy of renaturation depends

upon DNA concentration

M wt=2.5 x 107

T7 Molar= 4.4x T4

M wt=1.1x 108

Molecular Biology David Freifelder © Molecular Biology of Gene Watson et al ©

 in venaturation
Hybridization parameters
kinetics
1 Concentration of cations
209 below Tm
9 Incubation temperature
3 DNA concentration complexity of DNA
4 size of the fragments

nite

experimentatontwithisatfin ah

Ei mangy
Reanneling

long DNA
For molecules
must I 1 108
less
Ty 00
2 5 107 more molecules
M wot
Tf 08 renaturefaster
Chan th DNA
because con
of more
 EEE
É

MEET
F

anti I
 Cot
Cary
Rate of renauration
CAILLATB

s s
KAJ CBI AI
flay Kt

I
cone
initial
FAI of LA

fE
f Fat
n
Where co is initial concof basepair
in south and in each
N Number of base Dna complexity
se e
unique
C0t curve

1
f=
1+C0tk/x
f=fraction of unpaired strand
x= number of unique sequence
C0= initial con of base-pairs in solution
t=time
k=second order rate constant

When f=0.5
C0t1/2 =x/k

For a given set of conditions C0t½ depends upon the complexity of the DNA (x)
When k is rate constant (single strand collision)
 C-Value Paradox

human genome
is 650x
larger
E coli
than yet
encodes only N 5 h x
as
many structural
genes
 DNA Topology
(cccDNA) covalently closed circular DNA: The two ends are covalently linked to form a
circular DNA molecule (topologically constrained)
Linking number (Lk)
Interwound The number of times one strand have to
be passed through the other strand in
order to completely separate the two
strands. (it is always an Integer)

Lk=Tw +Wr
(Twist) (Writhe)

cccDNA free of supercoiling

(relaxed form) is denoted as LkO

Linking differences
Measures the extent of supercoiling

Lk – LkO=DLk
Negative supercoiled If Lk <LkO and DLk <0
Positive supercoiled If Lk >LkO and DLk >0
32-36= -4

Superhelical density of DNA

Molecular Biology of Gene Watson et al © s=DLk/LkO ~0.06
 Significances of DNA super-coiling
q The DNA super coil stores free energy that require strand separation, such as
DNA replication and transcription.
q Generally negative super-coiling is observed in Pro and Eukaryotic cells.
However, positive super-coiling was also found in thermophilic microorganism,
which keep their DNA from denaturation at elevated temp.
q Wrapping of DNA around nucleosome also induce negative supercoiling.
q Prokaryotes have a special type I topoisomerase known as DNA gyrase that
introduces negative supercoils and facilitate DNA supercoiling.

Ethidium increases Wr

Molecular Biology of Gene Watson et al ©

 Topoisomerase

Type I Type II

Type I topoisomerases change the

linking number of DNA in steps of one.
Topoisomerases can both catenate and
decatenate circular DNA molecules.
Molecular Biology of Gene Watson et al ©
 Topoisomerase-I in action

Molecular Biology of Gene Watson et al ©