DNA and RNA structure The four nucleotide subunits of DNA and RNA
N1 (Thymine and Cytosine)
N9 (Guanine and Adenine)
Organizzazione dei componenti del DNA. 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.
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
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
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)
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 B-form). Hybrids DNA-RNA also probably lie in the in the A-form.
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 cross-linking 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).
Psoralen photobinding (1987) Psoralen forms interstrand crosslinks photobinding to the 5’ TA of the 26 bp Z-DNA-forming sequence GAATT(CG)6TA(CG)6AATTC when this region is in the B-DNA but not when it exists as Z-DNA.
Exo III digests DNA from a 3’-OH
Restriction-modification assay (1987) (CG)13AATT(CG)13
Methylation prevents digestion by the corresponding endonuclease.
DNA is not restricted
DNA is restricted
Sequences that can be methylated, such EcoRI site GAATTC, require that the DNA exists in the B conformation for the methylase to act.
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)
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
The biology of triple-stranded DNA Intramolecular triplexes and gene regulation
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.
H-DNA may provide an entry site for RNA polymerase
C) Intramolecular triplexes may act as replication terminators
Genetic recombination involves an intramolecular triplex