Many naturally occurring DNA molecules are circular, with no free 5′ or 3′ end. Due to the polarity of the strands of the DNA double helix, the 5′ end of one strand can only join its own 3′ end to covalently close a circle. Thus, circular, double-stranded DNA is essentially two circles of single-stranded DNA twisted around
H
Figure 2.13 Hoogsteen base pairs. (A) AT, and (B) GC pairs. Symbols + and − are partial charges. Hydrogen bonds are represented by dotted lines. For simplicity, not all hydrogens are shown.
each other. Such circular DNA molecules often become overwound or underwound, with respect to the number of complete turns of the DNA double helix. This DNA can then become supercoiled (under torsional stress). Supercoils are a twisted, three-dimensional structure which is more favorable energetically.
Supercoiling of DNA can be visualized by electron microscopy and occurs both in vivo and in vitro.
Supercoiling of DNA
Consider a double-stranded linear DNA molecule of 10 complete turns (or twists, T= 10) with 10.5 bp/turn (Fig. 2.14). If the ends of the DNA molecule are sealed together, the result is an energetically relaxed circle that lies flat. Since each chain is seen to cross the other 10 times, this relaxed circle has a linking number (L) of 10. But, if the double helix is underwound by one full turn to the left and then the ends are sealed together, the result is a strained circle with 11.67 bp/turn, where L= 9 and T = 9. Once the two ends become covalently linked, the linking number cannot change. Generally, changes in the average number of base pairs per turn of the double helix will be counteracted by the formation of an appropriate number of supercoils in the opposite direction. In this example, one negative (left-handed) supercoil is introduced spontaneously, re-establishing the total number of original “turns” of the helix (T= 10, L = 9).
Overtwisting of the double helix usually leads to positive (right-handed) supercoiling. For example, if the double helix is overwound by one full turn to the right and then the ends are sealed together, the result is a
Linear DNA
10.5 bp/turn T=10
10.5 bp/turn T=10 L=10
Relaxed circle
Relaxed circle
Relaxed circle Cruciform structure
Partial denaturation Seal ends
together
Twist to the left one turn;
Seal ends together
Strained circle
Negative supercoil 11.67 bp/turn
T=9 L=9
T=10
10.5 bp/turn L=9
3′ 5′
3′ 5′ Figure 2.14 DNA supercoiling. A linear
DNA molecule of 10 complete turns (or twists, T= 10) is assumed to have 10.5 bp/turn, as is usual for B-DNA in solution.
The ends of the DNA molecule can be sealed together to make a relaxed circle. This relaxed circle has a linking number of L= 10. But, if the double helix is underwound by one full turn to the left and then the ends are sealed together, the result is a strained circle with 11.67 bp/turn, where L= 9 and T = 9.
One negative (left-handed) supercoil is introduced spontaneously, re-establishing the total number of original turns of the helix (T= 10, L = 9, 10.5 bp/turn). Upon partial denaturation, supercoiled DNA may convert to its unsupercoiled relaxed form. The single-stranded area may then convert to a more stable cruciform structure if there are inverted homologous sequences in the denatured regions (see Fig. 2.12).
strained circle with 9.5 bp/turn, where L= 11, T = 11. The introduction of one positive (right-handed) supercoil restores the total number of original turns of the helix (T= 10, L = 11).
The supercoiled state is inherently less stable than relaxed DNA. The stress present within supercoiled DNA molecules sometimes leads to localized denaturation, in which the complementary strands come apart in a short section. This has important implications for cellular processes such as replication and transcription.
Topoisomerases relax supercoiled DNA
Forms of DNA that have the same sequence yet differ in their linkage number are referred to as topological isomers (topoisomers). Topoisomers can be visualized by their differing mobilities when separated by gel electrophoresis (Fig. 2.15) (see Tool box 8.6 for methods). Topoisomerases are highly conserved enzymes that convert (isomerize) one topoisomer of DNA to another. They do so by changing the linking number (L). DNA topoisomerases fall into two major categories, type I and type II. The two types can be further subdivided into four subfamilies: IA, IB, IIA, and IIB. So far, at least five different topoisomerases have been reported to be present in higher eukaryotes, including humans (Table 2.3). The first topoisomerase was
1 2 3 4 5
Figure 2.15 Relaxation of supercoiled plasmid DNA by topoisomerase I.
Lane 1: Relaxed and supercoiled topoisomers of pUC18 plasmid DNA. Lanes 2–6: pUC18 plasmid DNA after treatment with 2, 4, 6, or 8 units of topoisomerase I, respectively, for 45 minutes at 37°C. DNA samples were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide (see Tool box 8.6 for methods). The speed with which the DNA molecules migrate increases as the number of superhelical turns increases.
Table 2.3 Human DNA topoisomerases.
DNA
topoisomerase I
IIIa
IIIb IIa
IIb
dsb, double-stranded break in DNA; ssb, single-stranded break in DNA.
Structural role
Relax both negatively and positively supercoiled DNA
Relax only negatively supercoiled DNA
Relax only negatively supercoiled DNA
Relax both positively and negatively supercoiled DNA Facilitate unknotting or decatenation of entangled DNA
Relax both positively and negatively supercoiled DNA Facilitate unknotting or decatenation of entangled DNA
Function Replication Transcription Recombination Recombination Transcription of ribosomal RNA genes Recombination
Chromosome condensation Chromosome segregation Replication
Not well defined Type
IB
IA
IA IIA
IIA
DNA cleavage ssb
ssb
ssb dsb
dsb
discovered by Jim Wang, while he was looking for the enzyme that relieves the supercoils that form during DNA replication (see Section 6.6).
Type I topoisomerases are proficient at relaxing supercoiled DNA. They do not require the energy of ATP. Type IA can only relax negative supercoils, while type IB can relax both negative and positive supercoils. They act by forming a transient single-stranded break in the DNA (cleavage of a phosphodiester bond between adjacent nucleotides) and, while winding the broken ends, pass the other strand through the break. Topoisomerases do not create free ends, but instead become themselves covalently attached to one of the two broken ends of the DNA (which one depends on the specific enzyme) (Fig. 2.16). The nick is then sealed, after relaxation increases the linking number by one.
Type II topoisomerases are usually ATP-dependent. They form transient double-stranded breaks in the double helix and pass another double helix through the temporary gap or DNA-linked “protein gate.” They are proficient in relaxing both negatively and positively supercoiled DNA and, in addition, can unknot or decatenate entangled DNA molecules (catenation is the interlocking of circles of DNA like links in a chain).
Prokaryotic topoisomerase II (sometimes called gyrase) has the special property of being able to introduce negative supercoils.
Both type I and type II topoisomerases play important roles in many cellular processes, including chromosome condensation and segregation, DNA replication, gene transcription, and recombination (Disease box 2.2).
Figure 2.16 Mechanism of action of a type I topoisomerase. The enzyme binds to a circular DNA molecule with one negative supercoil (see Fig. 2.14) and unwinds the double helix. It nicks one strand and prevents free rotation of the helix by remaining bound to each broken end. The 5′ broken end is covalently attached to the amino acid tyrosine (see inset), and the 3′ end is noncovalently bound to another region of the enzyme. The enzyme passes the unbroken strand of DNA through the break and ligates the cut ends, thereby increasing the linking number of the DNA by one. The enzyme falls away and the strands renature, leaving a relaxed circle. Inset:
in the strand breakage reaction by the topoisomerase, the oxygen of the tyrosine hydroxyl group in the active site of the enzyme attacks a DNA phosphorus, forming a covalent phosphotyrosine link between the DNA and the enzyme, and breaking a DNA phosphodiester bond at the same time.
Rejoining of the DNA strand occurs by the reverse. The oxygen of the free DNA 3′-OH group attacks the phosphorus of the
phosphotyrosine link, breaking the covalent bond between the protein and DNA, and reforming the phosphodiester bond between adjacent nucleotides in the DNA chain.
What is the significance of supercoiling in vivo?
Virtually all DNA within both prokaryotic and eukaryotic cells exists in the negative supercoiled state. If these domains are unrestrained (not supercoiled around DNA-binding proteins), there is an equilibrium between tension and unwinding of the helix. If the supercoils are restrained with proteins, they are stabilized by the energy of interaction between the proteins and the DNA. Experimental evidence suggests that DNA supercoiling plays an important role in many genetic processes, such as replication, transcription, and recombination.
Negative supercoiling puts energy into DNA. Underwinding makes it easier to pull the two strands of the double helix apart. Therefore, negative supercoiling makes it easier to open replication origins and gene promoters. The potential energy in the supercoils also promotes formation of unusual DNA secondary structures, like cruciforms (see Figs 2.12 and 2.14). In addition, it is possible that a B-DNA→ Z-DNA transition is triggered by increased negative supercoiling. This is because switching a portion of the DNA from a right-handed to left-handed helix releases the strain imposed by the negative supercoils, since the twist (base pairs per turn) in a portion of the DNA has been reversed. Positive supercoiling occurs ahead of replication forks and transcription complexes. Positive supercoiling makes it much harder to open the double helix and therefore blocks essential DNA processes.
Supercoiled DNA is a well-characterized feature of the circular genome of some small viruses, for example, bacteriophage PM2. The genome of this bacteriophage can exist as a relaxed circle (covalent closure) or a supercoiled circle, which has a twisted appearance. This is the native form in vivo (Fig. 2.17). In bacteriophages it has been shown that relaxed circular DNA correlates with reduced activity in replication and transcription, whereas negative supercoiling leads to increased activity in replication and transcription.
Similarly, bacteria have large, circular genomes that can form independent DNA loop domains. A protein complex holds each domain in place to form “subcircles” with an average size of ~40 kb. These domains may form supercoiled structures of importance for replication and transcription. Supercoiling also greatly facilitates chromosome condensation in bacteria.
Topoisomerases are the targets for a number of important anticancer drugs. Topoisomerase II-targeted drugs are used in approximately half of all chemotherapy regimens.
Topoisomerase I is the target for a number of promising agents, including camptothecin, a drug derived from the bark of Camptotheca acuminata, a Chinese yew tree.
Camptothecin was first isolated and found to kill certain types of cancer cells in 1966 by Dr Monroe E. Wall and Dr Mansukh C. Wani. It was not until 1985 that another group of researchers determined that camptothecin is a topoisomerase I poison. Now, camptothecin analogs are being used for the treatment of ovarian and colon cancer.
Topoisomerase-targeted anticancer drugs act in one of two ways, either as an inhibitor of at least one step in the catalytic cycle, or as poisons. Breaks created by topoisomerases are normally transient in nature (see Fig. 2.16). When the anticancer drug acts as a poison, the
broken DNA is trapped as a stable intermediate bound to topoisomerase. There is a high likelihood of this being converted to a permanent break in the genome.
These drugs preferentially target cancer cells because cancer cells are rapidly growing and usually contain higher levels of topoisomerases than slower growing cells. Due to their mode of action, the higher the cellular level of topisomerases, the more lethal these drugs become. In addition, cancer cells often have impaired DNA repair pathways, so they are more susceptible to the effects of DNA-damaging agents. Like all chemotherapeutic agents, drugs targeted to topoisomerases also affect normal fast growing cells, such as white blood cells, the lining of the gastointestinal tract, and hair follicles. This is why cancer patients undergoing chemotherapy often experience nausea, diarrhea, and hair loss, and are susceptible to infections.