The topology of nucleic acids Relevant aspects: The number of bases per turn is not fixed, but can be adjusted depending on the circumstances. The double helix does not exist as a long straight rod, but is coiled in space to fit into the dimensions of the cell. This additional level of organization (DNA supercoiling) places the molecule under stress in such a way that its own structure is affected. Discontinuities in the structure (bends). For DNA to be replicated or expressed the strands of the double helix must separate. DNA can be denatured and renatured Melting temperature (Tm) Hypochromic effect The A 260 nm of DNA is 40% less than the adsorption of a mixture of free nucleotides of the same composition. The Tm usually lies in a range of 85-95°C The Tm increases 0,4°C for every 1% increase in GC content.

Single-stranded nucleic acids may have secondary structure There are two common ways to perform hybridization: liquid or filter hybridization. Liquid hybridization Single-stranded nucleic acids may have secondary structure Stability of double helix results from: 1) Base pairing 2) Base stacking Stem-loop Gemma Ansa Pseudo-nodi

Inverted repeats and secondary structure Secondary structures of fMet-tRNA and 16S rRNA Inverted repeats and secondary structure Inverted repeats  two copies of an identical sequence present in a reverse orientation. 5’ GGTTCNNNGAACC 3’ 3’ CCAAGNNNCTTGG 5’ palindrome 5’ GGTTCGAACC 3’ 3’ CCAAGCTTGG 5’

Free energy DG total= DGi +  DGx +  DGu DGi is the free energy for initiation of a double helix (+3,4 kcal/mol). DGx is the free energy released in the formation of each base pairing (A/U pairs have values between -0,9 and -1,1 - doublets containing A/U and G/C pairs vary between -1,7 and -2,3 - doublets containing only G/C pairs vary from -2 to - 3,4 kcal/mol). DGu is the free energy to hold bases in an unpaired state so it is positive.

Closed DNA can be supercoiled Different topological conformations (topoisomers) of DNA molecule Supercoils are introduced into DNA when a duplex is twisted in space around its own axis. 1) Negative supercoiling  in the opposite direction from the clockwise turns of the right-handed double helix. 2) Positive supercoiling  in the same direction as the intrinsic winding of the double helix. Superhelical density is based on the concept of linking number which specifies the number of times that the two strands of the double helix of a closed molecule cross each other. The linking number cannot change unless the phosphodiester backbone is broken by chemical or enzymatic cleavage. L = T + W Twisting number (T) = it represents the total helical turns of the duplex. For a relaxed closed circular DNA, T is the total number of base pairs divided by the the number of base pairs per turn. Writhing number (W) = it represents the turning of the axis of the duplex in space and corresponds to the intuitive concept of supercoiling (for a relaxed molecule W=0).

In relaxed DNA L = T = 20 and W = 0 Introduction of negative supercoiling results in a change of writhe (W = -1). Since L is invariant in the absence of topoisomerase action (210  10,5 = 20) twist must change by + 1 (T = 21). In relaxed DNA L = T = 20 and W = 0 The linking number of relaxed DNA, Lo is defined as Lo = N/10,5 where N is the number of base pairs in the DNA molecule Linking number  = -DL = L < Lo = introduction of negative supercoiling Linking number  = +DL = L > Lo = decrease of negative supercoiling Sigma  = L/Lo superhelical density

Negatively supercoiled DNA A. Relaxed DNA. B. The Linking number has decreased by 2, L=18 (two helical turns have been removed by the action of a topoisomerase). This will change: the rotation per residue (Twist) from 34,3° (20 turns x 360°/210 bp) to 30,8 (18 turns x 360°/210 bp) the number of base per turn from 10,5 (210 bp 20 turns) to 11,7 (210 bp 18 turns) Energetically, this represents an unfavorable winding of the DNA double helix. C. Introduction of supercoils. 21 bp unwinding Denaturation is thermodynamically more favorable in negative supercoiled DNA than in relaxed or positively supercoiled DNA.

Twin-domain model (Liu and Wang 1987). Since positively supercoiled plasmid DNA could be purified from E. coli cells treated with novobiocin, an antibiotic that inhibits the activity of DNA gyrase (this enzyme is is able to introduce negative supercoils into DNA), it has been proposed that the formation of twin supercoiled domains occurs during transcription. The topoisomerase I of E. coli relaxes negatively supercoiled DNA. As the RNA transcript becomes large and in bacteria cells becomes bound to ribosomes, rotation around the DNA would difficult as well as the rotation of DNA itself.

Titration of supercoils with intercalating molecules About 12-13 molecules of Et-Br remove one turn of DNA double helix. A molecule of Et-BR reduces the normal rotation per residue (Twist) from 36° to 10°. As DNA becomes negatively or positively supercoiled, the molecules becomes much more compact and sediment more rapidly than relaxed DNA. Sedimentation in Ethidium Bromide sucrose gradients If negatively supercoiled DNA is sedimented in sucrose gradients containing an increasing concentration of Et-Br, then the sedimentation coefficient (s) will decrease. A minimum value is reached at the equivalent point where sufficient dye is bound to remove all negative supercoils (W = 0). On addition of more intercalator, s increases as positive supercoils are introduced into DNA. high low

Analysis of topoisomers distribution by chloroquine agarose gel The intercalation of the drug results in unwinding of the DNA and decreasing of DNA density. The unwinding relaxes negative supercoils. Standard agarose gel fails to resolve individual DNA topoisomers with increasing number of supercoils. Chloroquine is added to remove the excess of supercoils in order to change the the naturally occurring topoisomers into forms that will be in the range of gel resolution (0-15). Negative supercoiled DNA Relaxed DNA Positive supercoiled DNA Depleted cells Topoisomer distribution of the 4,4 Kb plasmid derived from pBR322 Wild type HU HNS HU HNS IHF DNA migration Relaxed DNA Supercoiled DNA