DNA must be highly condensed to fit within the confines of a cell. The double helix is constantly manipulated by cellular machinery for processes like copying genetic information and reading genes. To manage the physical stresses that arise from this manipulation, the cell relies on a class of enzymes called DNA topoisomerases. These molecular machines maintain the structural integrity and accessibility of the genetic material during all major cellular transactions.
Understanding DNA Supercoiling
DNA supercoiling describes the over- or under-winding of the double helix relative to its relaxed state, creating torsional stress. This topological problem is acute because chromosomal DNA ends are either constrained or joined in a circle, preventing free rotation.
When enzymes like helicase separate the two strands during replication, the DNA ahead of the separation point twists more tightly. This overtightening creates positive supercoiling, which increases torsional stress and can block the progression of separation machinery. Conversely, under-winding the helix introduces negative supercoiling, which helps prepare the DNA for strand separation. The accumulation of supercoils, particularly the positive variety, is the fundamental structural problem that topoisomerases solve.
The Core Action: Managing Torsional Stress
The primary function of topoisomerase is to relieve torsional stress, or supercoiling, by altering the DNA’s topological state. This action is distinct from helicase, which actively separates the two DNA strands, or “unwinds” the double helix. Topoisomerases act as molecular strain relievers, managing the tertiary structure of the molecule without separating the strands.
The mechanism involves a transient and reversible break in the DNA backbone, allowing the enzyme to change the overall winding without permanent damage. The enzyme cuts one or both strands, holds the ends, and allows the DNA to rotate or permits a separate segment of DNA to pass through the created gap. Once stress is relieved, the enzyme re-ligates the phosphodiester backbone, seamlessly repairing the break. This process changes the linking number of the DNA, a value quantifying the intertwining of the two strands, thereby managing supercoils.
Relieving supercoiling reduces the strain resulting from the strands being too tightly or loosely coiled. By cutting and rejoining the DNA backbone, topoisomerases change the geometry of the helix, allowing the torsional pressure created by cellular activity to dissipate. Without this precise action, torsional stress would cause replication or transcription machinery to stall.
Topoisomerase Types and Specific Mechanisms
Topoisomerases are categorized into two major types based on the number of DNA strands they transiently cut.
Type I Topoisomerases
Type I topoisomerases are monomers that create a transient single-strand break in the DNA duplex. This single break allows the uncut strand to swivel or rotate around the phosphodiester bond of the broken strand, relaxing the supercoils.
The Type I mechanism changes the linking number of the DNA by increments of one, removing one coil at a time. Most Type I enzymes do not require cellular energy (ATP) because the energy stored in the supercoiled DNA powers the rotation and re-ligation. Eukaryotic Type IB topoisomerases relax both positive and negative supercoils, while many bacterial Type IA enzymes relax only negative supercoils.
Type II Topoisomerases
Type II topoisomerases are generally dimers or tetramers that require a double-strand break in the DNA. These enzymes use a sophisticated strand-passage mechanism. They bind to one double-stranded DNA segment, create a break, and then actively pass a second, intact double-stranded segment through the temporary gap. This process is energy-dependent, requiring the hydrolysis of ATP to fuel the necessary conformational changes.
The action of Type II enzymes changes the linking number by increments of two, making them efficient at resolving intertwined DNA segments. Bacterial DNA gyrase is a notable Type II enzyme that actively introduces negative supercoils into DNA, maintaining the correct tension in the bacterial genome. All Type II enzymes are also essential for untangling linked DNA molecules, known as decatenation.
Cellular Roles in Genome Maintenance
Topoisomerases play a pervasive role in genome maintenance, ensuring that packaged DNA remains accessible. During DNA replication, the movement of the helicase enzyme creates a build-up of positive supercoils ahead of the replication fork. Type I and Type II topoisomerases work together to quickly relax this torsional stress, preventing the replication machinery from jamming.
The enzymes are also crucial during gene expression, as the movement of the transcription machinery creates supercoils both ahead of and behind the complex. Topoisomerases constantly alleviate this strain to allow genes to be read. Furthermore, Type II topoisomerases untangle the newly duplicated daughter chromosomes during the final stages of cell division, ensuring correct segregation into the two new cells.
The reliance of proliferating cells on these enzymes has made them a significant target in medicine. Chemotherapy drugs, such as irinotecan and etoposide, inhibit human topoisomerases by trapping the enzyme-DNA complex after the cut but before re-ligation. This action converts the enzyme into a DNA-damaging agent, leading to cell death in rapidly dividing cancer cells. Similarly, antibiotics like fluoroquinolones target and inhibit the bacterial Type II topoisomerases, DNA gyrase and topoisomerase IV, halting bacterial replication.