Why Is DNA Like a Plumber’s Snake?

Kouzine F, Liu J, Sanford S, Chung HJ, and Levens D. The dynamic response of upstream DNA to transcription-generated torsional stress. Nat Struct Mol Biol 11: 1092–100, 2004.

he double helix must be untwisted to decode or copy the genetic information embedded in DNA sequences. As one strand is locally rotated about the other, torsional stress is inevitably generated. In topological domains where the total number of helical turns is fixed, which may occur by a number of means such as protein-protein interactions between DNA-bound factors, torsional stress accumulates as DNA is unwound, causing the double helix to coil back upon itself to form “supercoils” (Figure 1, part A). Because double-stranded DNA is intrinsically very stiff, unless dissipated, the supercoiling forces stored in DNA may rise to levels that alter its structure or impede the enzymatic machineries that conduct genetic business (i.e., transcription, replication, recombination, and repair). Therefore, special enzymes, termed “topoisomerases,” cut one or both DNA strands to allow rotations that release torsional stress and then reseal the cuts (Figure 1, part A, middle).

Figure 1. A) When a DNA fiber is wrapped around itself, with its ends restrained (in this case, the ends are fixed to each other forming a circle), supercoils are formed and trap torsional stress (top). The only way to remove the stress is to break the DNA and let the ends counter-rotate (middle). We wondered what happens dynamically if stress is applied to an open DNA fiber. If the DNA is rigid enough, the whole molecule would rotate as a unit and no supercoils would form, or if the DNA is flexible enough, the whole fiber would writhe around itself in response to applied torque (bottom). B) Linear, open molecules with divergent promoters were transcribed in vitro; concurrent recombination between loxP sites bracketing the interposed DNA, which was excised as closed circles, trapped any supercoils residing in or transiting through the interposed segment at the instant of recombination. Transcription generates torque as the double helical template is threaded through the RNA polymerase active site. Without transcription (Trx), no stress would be captured; the number of transient supercoils captured was expected to reveal how rigid or flexible the DNA was. C) A sample result: ongoing transcription (+) traps a large number of supercoils. Without transcription (–) very few supercoils are trapped. D) Factors recognizing dynamic changes in DNA structure resulting from transcriptional torque provide the necessary effector components to construct a molecular “cruise control.” FUSE, far upstream element; FBP, FUSE binding protein; FIR, FBP interacting repressor; loxP, target sequences for the site-specific Cre recombinase. T3 and T7 indicate bacteriophage T3 and T7 promoters.

Although supercoiling forces often modify gene expression in prokaryotes, in metazoans, the capacity of vast stretches of non-coding DNA to absorb this stress and abundant topoisomerase activity have been presumed to mitigate the influence of supercoiling on gene regulation. Moreover, attempts to measure the stable level of supercoils per unit length of DNA (the superhelical density, σ) suggest that torsional stress does not accumulate to high levels in the DNA of higher eukaryotes.

We wondered, however, what does DNA (in this case linear DNA that cannot hold onto supercoils) look like dynamically while it is being transcribed? Does it writhe like a plumber’s snake being whipped about, or do the supercoils run off the ends of the DNA so rapidly that the template is relaxed and unstressed (Figure 1, part A, bottom)?

The experimental problem was to trap the evanescent stresses propagating from an activated promoter in linear DNA. These dynamic supercoils had to be captured and preserved during transcription for future study, because active RNA polymerase translocating along the template was the kinetic engine cranking the DNA. The trick was to convert the dynamic supercoils into stable, conventional supercoils. To accomplish this, a 1-kilobase (kb) (100 double helical turns) segment of DNA was interposed between two similarly oriented loxP sites, target sequences for the site-specific Cre-recombinase (Figure 1, part B). This loxP-bracketed segment was in turn placed upstream of a single phage RNA polymerase promoter or was inserted between two divergently transcribed phage promoters, all in linear DNA fragments. With the latter arrangement, the dynamic supercoils from each promoter would be expected to be mutually reinforcing. Upon addition of Cre, site-specific recombination between the loxP sites was expected to excise a 1-kb, covalently closed DNA circle, trapping any supercoils residing in or transiting through the segment at the instant of recombination (Figure 1, part B). Two-dimensional electrophoresis of the 1-kb circles recovered from these reactions to separate the DNA rings into a series of spots, each differing from its neighbor by a single supercoil, promised to give an accurate accounting of dynamic supercoils generated during transcription (Figure 1, part C).

In the absence of transcription, recombination trapped no more than the two supercoils explained by thermal wriggling of DNA. As the transcription intensity was increased, the circles trapped more and more supercoils. As many as 14 supercoils were captured in the 1-kb segment; σ = 0.14, an incredibly high number (Figure 1, part C). What are the biological consequences of σ rising transiently to such a high level?

At high levels of σ, DNA melts at susceptible sites. This melting does not occur gradually, but at critical thresholds, the susceptible segments, “soft spots” in the DNA, buckle. In fact, from 2-dimensional gel electrophoresis, chemical modification, and nuclease hypersensitivity assays, we show that the far upstream element (FUSE) from the human c-myc gene pops open during transcription initiated at downstream promoters. Depending on the σ, melted FUSE binds an activator, the FUSE binding protein (FBP), and a repressor, the FBP interacting repressor (FIR). Thus, these proteins superimpose effector function on the stress-sensor properties of FUSE and, in principle, create a mechanical device for the real-time regulation of transcription (Figure 1, part D). Real-time regulation is likely to be especially important for genes yielding short half-life, low abundance transcripts, such as from c-myc and perhaps other protooncogenes, tumor suppressors, and cell cycle regulators.

Focal melting of DNA may have several other important consequences: (1) Melted DNA is much more flexible than duplex, so a melted segment may help to juxtapose widely separated elements and their associated factors. (2) Dynamic supercoiling may energetically assist chromatin remodeling and modification. (3) Propagation of dynamic supercoils from one gene to another, in principle, allows the activity of one gene to modulate directly and immediately the activity of a closely situated promoter. Knowledge of the transmission of mechanical forces through DNA may help us to understand chromosome architecture and to devise strategies for the precise control of genetic processes.

Fedor Kouzine, PhD
Visiting Fellow
kouzinef@mail.nih.gov

David Levens, MD, PhD
Senior Investigator
Laboratory of Pathology
NCI-Bethesda, Bldg. 10/Rm. 2N105
Tel: 301-496-2176
Fax: 301- 594-5227
levens@helix.nih.gov