Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432	Biochemistry	Spring 2002
Lecture Notes:: 27 February	© R. Paselk 2002
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DNA Structure and Folding

DNA exists as a double-stranded, anti-parallel, double helix (both strands wrap together, like a ribbon, around a common axis). There are three different DNA structures, as seen in Figures 23-2 & 23-3, pp 728-9 of your text. Their "ideal" properties are:

• A-DNA - Occurs in DNA when dehydrated. Closely approximates the conformation of RNA helices.
  • Right-handed, approximately 26Å diameter, double-helix, with 11 base-pairs/turn giving a pitch of 28Å.
  • The planar bases are tilted about 20° to the axis.
  • The larger diameter than seen in B-DNA leaves a hole in the center, and unlike B-DNA, the axis does not pass through the base-pairs.
• B-DNA - Most stable form, predominating in biological organisms.
  • Right-handed, approximately 20Å diameter, double-helix, with 10 base-pairs/turn giving a pitch of 34Å.
  • Only two types of base-pair, AT & GC, can be accomodated in this structure without distortion. Because these two base-pairs have the same C₁'-C₁' distance they can be interchanged along the helix with no change in the overall structure.
  • The planar bases are nearly perpendicular to the helix axis and partially stacked.
  • The bases lie in the core of the helix, nearly inaccesible to solvent, while the sugar-phosphate backbones wrap around the outside, forming the major and minor grooves.
• Z-DNA - May occur in short stretches of DNA acting as a regulatory switching element, but biological existance uncertain.
  • Left-handed, approximately18Å diameter, double helix, with 12 base-pairs/turn giving a pitch of 45Å.

As demonstrated in the Meselson and Stahl experiment, DNA replication is semi-conservative (see Figure 24-1, p 773 of yoiur text).

The Supercoiling of DNA

DNA in vivo is supercoiled in various ways. Supercoiling allows DNA to take up a more compact form, and is a necessary consequence of replication and transcription.

So what is supercoiling? Supercoiling means that the helix is itself wrapped into a higher level coil, it is super-twisted or has super-helicity.

We will discuss supercoiling in terms of topology, the study of propwerties of geometric forms, and the transformations that leave them invarient in certain properties (e.g. a tea cup and a donut are topologically the same in that they have a single surface penetrated by a single hole).

Super helix topology. The topology of the DNA helix/supehelix can be described by a simple equation:

where:

• L = the linking number = the number of times one strand crosses the other. Note that this is invarient to twists and distortions so long as neither strand is cut! Thus if the primary helix is unwrapped, then it must wrap into a supercoil (demo with belt or tubing model).
• T = twist = number of complex revolutions one strand makes around the duplex axis in the conformation being considered.
  • T is positive (+) for right-handed duplex turns.
  • T is negative (-) for left-handed duplex turns.
  • For B-DNA the helix is right-handed, so T = + = (# base-pairs)/10.4, where 10.4 is the observed number of turns for aqueous B-DNA.
• W = writhing number = the number of turns the duplex axis makes around the superhelix axis in the conformation being considered. This is a measure of superhelicity.

So let's look at DNA interms of topology. The best information is from viruses and plasmids - both are small enough to work with in whole form.

Bacteria, plasmids, mitochondria and viruses usually have circular DNA, or in the case of viruses, the DNA becomes circularized after injection into the host.

• Note the variable amounts of supercoiling in the micrographs of these viral genomes. [overhead 28-33] (Figure 23-7, p 732)
• A twisted telephone cord, or a belt can demonstrate the differences between W (writhing) and T (twisting). [overhead 28-34] (Figure 23-8, p 733)
• For a closed circular piece of DNA note that the value of L is fixed, thus if the helix is overwound (a twist is added, T + 1), then it must supercoil in a negative sense (W must go negative by -1). In the example L = 9 and T changes from 9 to 10, thus forcing W to change from 0 to -1 (or vice versa). [overhead 28-36] (Figure 23-7, p 733)
  • Note that L is strictly only fixed for a closed circle, however, if the ends of a linear piece of DNA are fixed, as they effectively are by the bulk of a eukaryotic chromosome, then L will be effectively fixed for any relatively short sectin in the chromosomes interior.
• Experimentally the relationships of twist and writhing are observed in short closed circular genomes when the dye ethidium bromide (EtBr) is added incrementally. EtBr intercalates between bases in DNA, effectively forcing an unwinding of the helix (T decreases). Normal DNA is strongly negatively supercoiled, thus the addition of EtBr results in an incremental change of W from (-) to (+) as shown in the figure (inferred via centrifugation studies) [overhead 28-38] (Figure 23-7, p 734)

Topoisomerases and the Control of Supercoiling in DNA

Supercoiled DNA can relax by nicking (single strand cleavage such as by DNAaseI) creating a "swivel."

DNA must have the proper topological state for normal biological function (replication, recombination, transcription). Normal negative supercoiled state aids unwinding during replication and transcription.

Supercoiling is introduced and controlled by topoisomerases. These enzymes change the linking number (L) in DNA and thus its topology. Two main types:

• Topoisomerase I operates by making single strand breaks.
• Topoisomerase II operates by making double strand breaks.

Type I topoisomerases relax supercoiling incrementally (change L ±1) by reversibly catenating the DNA strands.

• Done by reversible formation of a tyrosine-phospho-DNA strand on the enzyme (prokaryotes: 5'P; eukaryotes: 3'P), thus maintaining the "high energy" bond of the DNA strand, so the DNA strand can be relinked without any requirement for ATP.

Type II topoisomerases or Gyrases, require an energy source:

• NAD in eukaryotes,
• ATP in prokaryotes.

These enzymes catalyze the stepwise negative supercoiling of DNA in prokaryotes (catalyze the relaxation only in the absense of ATP).

Eukaryotes only catalyze realaxation of supercoiling via other enzymes.

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Last modified 10 March 2002