Supercoiling Affects DNA Structure

Supercoiling places DNA under physical strain. This may lead to the appearance of alterations in DNA structure that serve to relieve the strain. Three of these alternate forms are cruciform structures; the left-handed double helix, or Z-DNA; and the triple helix, or H-DNA. All of these structures depend on certain special characteristics within the DNA sequence, as well as supercoiling stress.

catenane Structure in which two or more circles of DNA are interlocked cruciform structure Cross shaped structure in double stranded DNA (or RNA) formed from an inverted repeat

H-DNA A form of DNA consisting of a triple helix. Its formation is promoted by acid conditions and by runs of purine bases

Z-DNA An alternative form of DNA double helix with left-handed turns and 12 base pairs per turn

Supercoiling Affects DNA Structure 91

Negative electrode

Stained bands of DNA

Negative electrode

Stained bands of DNA

Movement of DNA

FIGURE 4.19 Separation of Supercoiled DNA by Electrophoresis

Movement of DNA

FIGURE 4.19 Separation of Supercoiled DNA by Electrophoresis

Supercoiled DNA molecules, all of identical sequence, were electophoresed to reveal multiple bands, with each band differing in the number of supercoils. The number of supercoils is shown beside the band. Zero refers to open circular DNA, which is not supercoiled at all.

Alternative helical structures are sometimes found for DNA, in addition to the common form, made famous by Watson and Crick and known as B-DNA.

The A-form helix is found in dsRNA or DNA/RNA hybrids. It has 11 bp per turn—one more than B-DNA.

Separation of Topoisomers by Electrophoresis

DNA molecules of different sizes are routinely separated by electrophoresis on agarose gels (see Ch. 21). The mobility of a DNA molecule in such a gel depends on both its molecular weight and its conformation. Heavier molecules travel more slowly but among molecules of the same molecular weight, those that are more compact move faster. Consequently, for a given DNA molecule, supercoiled cccDNA moves faster than open circular DNA, which in turn moves faster than linearized DNA.

For small circular molecules of DNA, it is even possible to separate topoi-somers with different numbers of superhelical twists. The more superhelical twists, the more compact the molecule is and the faster it moves in an elec-trophoretic field (Fig 4.19).

When the DNA gyrase of an E. coli strain containing small plasmids is inhibited, it is possible to isolate the plasmids and demonstrate that they are now positively supercoiled. Topoisomerase I continues to remove the excess negative supercoils but the DNA gyrase no longer cancels out the surplus positive super-coils, which therefore accumulate.

The cruciform (cross-like) structures are formed when the strands in a double stranded DNA palindrome are separated and formed into two stem and loop structures opposite each other (Fig. 4.20). The probability that cruciform structures will form increases with the level of negative supercoiling and the length of the inverted repeat. In practice, the four to eight base sequences recognized by most regulatory proteins and restriction enzymes are too short to yield stable cruciform structures. Palindromes of 15 to 20 base pairs will produce cruciform structures. Their existence can be demonstrated because they allow single strand specific nucleases to cut the double helix. (A nuclease is an enzyme that cuts nucleic acid strands; see Ch. 22) Cutting occurs within the small single stranded loop at the top of each hairpin. Cruciform structures partially straighten supercoiled DNA and so the molecule is not folded up as compactly and thus travels slower during gel electrophoresis.

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