DNA and RNA structure The four nucleotide subunits of DNA and RNA N 1 (Thymine and Cytosine) N 9 (Guanine and Adenine) DNA RNA
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 ~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° 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 alternative chemical forms of the bases TAUTOMERIZATION Transitions: Keto (C=O) form enol (C-O-H) form ( frequency 1: 104) Amine (-NH 2) 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 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 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
Psoralen photobinding (1987) B-DNA Exo III digests DNA from a 3’-OH Psoralen forms interstrand cross-links photobinding to the 5’ TA of the 26 bp Z-DNA-forming sequence GAATT(CG)6 TA(CG)6 AATTC when this region is in the B-DNA but not when it exists as Z-DNA. Restriction-modification assay (1987) (CG)13 AATT(CG)13 Methylation prevents digestion by the corresponding endonuclease. Sequences that can be methylated, such Eco. RI site GAATTC, require that the DNA exists in the B conformation for the methylase to act. DNA is not restricted DNA is restricted
Twist - Roll - Slide - Propeller Twist Local alterations of DNA structure T = 28° 40° R = +20° -10° pos. S = +2 A -1 A (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 p. H. 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
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