L> DNA Structure/TopologyBiol/Chem 5310Lecture: 24November 21, 2002Nucleic AcidsDNA Structure and Topology (Ch.23)Stability of the Double Helix2 Single strands 1 Double strandDG for this procedure should be negative, if the double helix is stableWhat accounts for the stcapacity of the duplex?This procedure deserve to be studied by raising temperature to induce"melting".Ionic toughness affects the stcapability of duplex DNA bereason of the phosphates. An boost in I, shields the negative charge of phosphates from each other. The duplex is more steady, the Tm (melting temperature) is higher.(Lower I, the Tm is reduced, duplex much less secure.)Let"s think about what accounts for the stcapability .The burying of the bases. But this is not the hydrophobic effect,as in proteins and also lipids. The bases are not ssuggest nonpolar,such as the alkyl chains of amino acids or lipids, or the phenylrings of Phe, Tyr.
AdenineGuanineCytosineThymine
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The Tm relies upon the base composition-it increases with GC content.This is not as a result of the additional hydrogen bond in G-C pairs, relativeto A-T pairs.As is true in proteins, such hydrogen bonds perform not contributea lot to thermodynamic stcapacity of folded proteins. (single strandbases will certainly make the very same variety of about indistinguishable hydrogenbonds in solution as in duplex DNA.)Also, low polarity solvents destabilize DNA. Such solvents would strengthen H-bonds. So other interactions must be weakened by such solvents.The formation of duplex DNA is recognized not to be entropy-driven, in contrast to the folding of proteins.The stcapacity of double-stranded DNA is thought to be because of especially favorable stacking interactions in between the base-pairs. These might involve induced-dipoles and also various other such points. The toughness of these interactions is highly-dependent upon the 2 base-pairs that communicate. Since tright here are 4 bases, tright here are 4 x 4 = 16 possible stacks of 2 base pairs (minus 6 that are precisely tantamount (e.g. AC is indistinguishable to GT)

, tbelow are 10). In basic, stacking interactions through a GC base pair are stronger than those with an AT bp. (watch Homework #10)

The melting of DNA can be detected as a rise in absorption at 260 nm, as the temperature is raised via the Tm. This is bereason single strand also base absorbs more than execute stacked bases. (Free nucleotides absorb even more.)This is concerned the complex digital interactions of the stacked bases. Remember they are all planar, and in van der waals call.The temperature of the midsuggest of the shift is the Tm.This is a participating phenomenon. The melting occurs duringa very small variety of temperature. That is because it is difficultfor the first few bases to dis-interact from the duplex. Once theduplex starts to unwind, it is a lot simpler for all the remainder ofthe bases to break free. (For noncooperative melting, view PolyA,Fig 23-21 p742, text )Supercoiled DNA: Tertiary Structure (See Guided Exploaration 22 on the CD-ROM)DNA of virsupplies, bacteria, mitochondria-frequently uncovered as circular, covalently-closed. Can be watched by electron microscopy. The additional coiling is "supercoiling".Such molecules have topological properties. Tright here is a number, L, the linking number, equal to the number of times that one strand also winds roughly the various other.L should a totality number. Since the strands are circular, travelingalengthy one strand also, one constantly returns exactly to the place onestarted from.L remains the same in the time of any type of actions entailing twisting orunwinding of the 2 DNA strands.L might change, only if one or both strands are damaged, and also reformedafter unwinding or twisting.Demonstrations:1) Rubber tubing as a version of duplex DNA. Breaking, twistingand also recreating generates supercoils as watched in EM"s.2) Strips of paper as a version of duplex DNA. Cutting apartthe "strands" after forming supercoils allows us tosee that one strand also is wrapped around the various other.Now take into consideration a circular DNA of 10,400 base pairs, planar, completelyserene with 10.4 bp/revolve.So tbelow are 10400/10.4 = 1000 transforms.This is "T", the twist, characterized as the number oftimes that one strand also winds around the various other in "Watson-Crick"create.In this instance, L = T = 1000.In general, L = T + Wwright here W is the wripoint number, the additional number of times that one strand winds about the various other. It is taken into consideration to be the number of supercoils. It can be negative or positive.Both T and also W require not be entirety numbers (just L have to be, ina circularly-closed molecule).If no bonds are broken during a procedure, L is solved, however bothT and W can change.Because DL = 0,DT = - DWDT and DWrequire not be entirety numbers.In right-handed DNA, e.g. A-form and also B-form, T is positive.In Z-create DNA, T is negative.So, both T and W have the right to be either positive or negative. DT and DW canalso be positive or negative.

4 More Points about Supercoiling1) A negatively-supercoiled DNA (typical situation) will bein equilibrium through a less-negatively supercoiled DNA that ispartly unwound. That is, as DW >0, DT As W increases, T decreases (L does not readjust unmuch less bonds are broken).The physiological definition of this is that in negatively supercoiled DNA, the duplex DNA deserve to be easily unwound to facilitate copying of the DNA design template (replication or transcription).2) Liclose to DNA (e.g. chromosomes of many organisms) deserve to alsoexhilittle bit supercoiling if the 2 ends are addressed by binding to a proteincomplicated. Model: telephone and cord.3) Nicking relaxes supercoiled DNA. Nicking is the breakingof one phosphodiester bond in one strand also of DNA. Supercoilingis lost because of rotation roughly single bonds on the unbrokenstrand also.4) Intercalating agenrts impact supercoiling, e.g. the DNA stain ethidium bromide. It fits in between stacked bases and also reasons local unwinding.

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Control of SupercoilingTopoisomerases are enzymes that transform one topolological stateor topoisomer to another. This is a readjust in L. These enzymesshould break bonds in the strands of DNA, and also then redevelop them.2 examples:1) Type I topoisomerases rise L by 1, by removing 1 negativesupercoil. The enzyme renders a single-strand break (a nick), unwindsthe helix one rotation, then reforms the second strand also. (Chimelink)2) Type II procaryotic topoisomerases decrease L by 2 (DL = -2). They break both strands in a duplex. The reactivity is thrust by ATP because it is otherwise unfavorable. It rises negative supercoiling. Proposed device in Fig.


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Last updated Wednesday, November 20, 2002