Base pairing

DNA and RNA structure
The four nucleotide subunits of DNA and RNA
N1 (Thymine
and Cytosine)
N9 (Guanine
and Adenine)
DNA
RNA
Legame glicosidico
Organizzazione
dei componenti
del DNA.
Legame fosfodiesterico
Esternamente è
mostrato lo
scheletro
(backbone)
zucchero-fosfato
ed al centro della
molecola sono
indicati i legami
idrogeno fra le
basi.
Nota
l’organizzazione
antiparallela dei
nucleotidi.
Legame idrogeno
Schema di lettura convenzionale:
5’- C A G T T- 3’
3’- G T C A A- 5’
Attenzione!! T T G A C NON è la sequenza di questo frammento di
DNA, e NEPPURE del filamento complementare
Il modello di Watson-Crick o DNA-B
~ 12 Å
~22 Å
Since the base pairs are attached asymmetrically to the backbone, one groove
between the strands is wider than the other. These are called the major and the
minor groove. Both grooves provide opportunities for base-specific interactions,
but the major groove is better suited for that task and more often observed as the
primary binding site for proteins.
12 Å
22 Å
Base pairing
240°
120°
120°
240°
The DNA double helix is stabilized by:
- hydrogen bonding (2-3 Kcal/mol)
- base stacking interactions (hydrophobic interactions and Van der Waals forces
between the planar rings of the aromatic bases)   4-15 Kcal/mol per dinucleotide
In addition to the traditional Watson-Crick configuration there are
alternative chemical forms of the bases  TAUTOMERIZATION
Transitions:
Keto (C=O) form  enol (C-O-H) form ( frequency 1: 104)
Amine (-NH2) form  imine (=NH) form (frequency 1: 104)
Keto-Enol
N
Amine-Imine
O
Keto-Enol
Bases can exist in ionized forms that change their
hydrogen bonding properties.
The C-A+ (protonated)
pair involves wobble
pairing
The C+ (protonated)-G
base pair involves
Hoogstein pairing
Mispairing errors during DNA replication that lead to mutation do not occur at the
high frequency one might expect given the possibilities for tautomeric shifts,
ionizations and wobble base pairing because bacterial and animal cells have
evolved elaborate mechanisms to deal with these mispairing:
- proofreading activity of DNA polymerase
- repair systems
I gruppi chimici all’interno del solco maggiore identificano le basi,
permettendo di distinguere una coppia A:T da una C:G ma anche A:T
da T:A e C:G da G:C.
Il solco minore non è così ricco di informazioni. E’ comunque
possibile discriminare una coppia A:T da una C:G.
A = accettore di legame idrogeno
M = gruppo metilico
D = donatore di legame idrogeno
H = idrogeno non polare
minor
groove
major
groove
major
groove
minor
groove
major
groove
The A-form (dehydrated form of DNA) is
biologically interesting because it is
probably very close to the conformation of
double-stranded regions of RNA (the 2’-OH
group prevents RNA from lying in the Bform). Hybrids DNA-RNA also probably
lie in the in the A-form.
minor
groove
Under certain conditions, a
stretch of G-C can convert
to Z-DNA, while the
regions on either sides
remain in the B-form.
The Z-DNA exists in vivo
Antibodies can distinguish the Z-form of DNA from the B-form (1982)
Following incubation of DNA with antibodies against Z-DNA and crosslinking reaction, samples were passed through a nitrocellulose filter (free DNA
fragments passed through the filter but not DNA-antibody complexes). Z-DNA
was retained. Samples were analyzed by electrophoresis on gel and compared
with control DNA not exposed to antibody (analysing missing, or reduced in
intensity, bands).
DNA fragments
Z-DNA
No antibody anti-Z-DNA
+ antibody anti-Z-DNA
filtration
electrophoresis on gel
Restriction-modification assay (1987)
(CG)13AATT(CG)13
DNA is not restricted
DNA is restricted
Methylation prevents digestion by the corresponding
endonuclease.
Sequences that can be methylated, such EcoRI site GAATTC,
require that the DNA exists in the B conformation for the methylase
to act.
The DNA methylase does not function on Z-DNA.
Twist - Roll - Slide - Propeller Twist
Local alterations
of DNA
structure
T = 28° 40°
R = +20°  -10°
pos.
S = +2 A  -1A
(conformazione a pale d’elica)
pos.
One complete helical turn having T=36°, showing the effects of
introducing uniform roll R or slide S at each step
R= 0° , S= 0 A
DNA-B
R=0° , S=2 A
R=12° , S=0 A
R=12° , S=2 A
DNA-A
Intramolecular Triplexes (H-DNA)
DNA containing repeated tracts of pyrimidine and purines
can form a three-stranded helix in response to negative
supercoiling or low pH.
The polypyrimidine strand
folds back and inserts
itself into the major
groove of the remaining
duplex.
Hoogsteen base pairs
Watson-Crick base pairs
3 filaments
The biology of triple-stranded DNA
Intramolecular triplexes and gene regulation
Activator

Repressor

A B-DNA binding protein
would bind to a gene and
facilitates the binding of RNA
polymerase. H-DNA results in
gene repression.
A B-DNA binding protein
may bind to the promoter
sequence and acts as repressor
preventing RNA polymerase
binding. H-DNA results in
gene activation.
SSB
H-DNA may provide an entry
site for RNA polymerase
C) Intramolecular triplexes
may act as replication
terminators
D)
Genetic
recombination
involves an
intramolecular
triplex