DNA within the human cell nucleus faces a constant challenge managing its immense length without becoming tangled. Processes like copying DNA and separating chromosomes generate immense topological stress, resulting in twisted, knotted, or interlinked strands. Human DNA Topoisomerase II is a molecular machine designed to resolve these complex topological problems by cutting and rejoining double-stranded DNA segments. This enzyme’s precise control over DNA structure is necessary for genome integrity and successful cell division.
Defining the Enzyme’s Structure
Topoisomerase II is classified as a Type II topoisomerase because it has the unique ability to cleave both strands of the DNA double helix simultaneously. The functional enzyme operates as a homodimer, meaning it is composed of two identical protein subunits working in concert. This complex structure requires energy to perform its function, utilizing the hydrolysis of Adenosine Triphosphate (ATP) to power its movements. The enzyme is organized into distinct domains: an N-terminal ATP-binding domain, a central DNA-gate domain where the DNA is cleaved, and a C-terminal domain. The human genome encodes two main isoforms: Topoisomerase II Alpha (TOP2A) and Topoisomerase II Beta (TOP2B), which share the same basic mechanism but have distinct cellular roles.
Mechanism of DNA Strand Passage
The enzyme resolves DNA tangles through the strand passage mechanism. This process begins when the enzyme binds to two separate DNA segments: the Gate (G) segment, which will be cut, and the Transported (T) segment, which will pass through the break. The binding of two ATP molecules causes the enzyme to clamp down, capturing the T-segment inside its structure.
This ATP-driven change cleaves the G-segment in both strands, creating a temporary, protein-bridged double-strand break. The enzyme forms a covalent bond with the 5’ end of each broken strand, preventing the DNA ends from floating away. The captured T-segment is then physically passed through the gap created by the broken G-segment.
Once the T-segment passes through, the enzyme religates the double-strand break in the G-segment, restoring its integrity. Finally, the hydrolysis of the bound ATP facilitates the opening of a lower C-gate, allowing the T-segment to be released. This cycle changes the topological state of the DNA by a unit of two, successfully unlinking or relaxing the molecule.
Topoisomerase II and the Cell Cycle
The activity of Topoisomerase II is precisely regulated to coincide with the most demanding stages of the cell division cycle. Topoisomerase II Alpha (TOP2A) is necessary for cell proliferation, as its expression is restricted primarily to the S (synthesis) and M (mitosis) phases. Its primary responsibility is to resolve the massive tangles, known as catenanes, that link the two newly replicated sister chromatids together. TOP2A is the only enzyme capable of decatenating these linkages, a process concentrated at the centromeric regions during metaphase. If TOP2A activity is blocked, sister chromatids cannot fully separate, leading to a failure of chromosome segregation and cell death. In contrast, Topoisomerase II Beta (TOP2B) is expressed constitutively in both dividing and non-dividing cells and is not required for sister chromatid separation. TOP2B primarily functions in processes like gene regulation and transcription.
Exploiting Topoisomerase II in Medicine
The high reliance of dividing cells on TOP2A has made the enzyme a prime target for a major class of chemotherapy drugs. These drugs are highly effective against cancers because cancer cells proliferate rapidly, leading to high expression and activity of TOP2A. Inhibitors of Topoisomerase II are categorized into two distinct classes based on their mechanism of action.
Topoisomerase II Poisons
The first and most commonly used class are Topoisomerase II poisons, which include frontline agents like etoposide and doxorubicin. These drugs stabilize the enzyme after it has created the double-strand break but before it can religate the DNA. This action traps the enzyme in a lethal complex with the broken DNA, resulting in permanent double-strand breaks that overwhelm the cell’s repair mechanisms and trigger programmed cell death.
Catalytic Inhibitors
The second class is known as catalytic inhibitors. These drugs prevent the enzyme from completing its entire cycle without creating lethal DNA breaks. Catalytic inhibitors, such as merbarone and ICRF compounds, often work by preventing the enzyme from binding to or hydrolyzing ATP, effectively stalling the strand passage process. While they do not cause immediate DNA damage like poisons, they are being investigated because they suppress cell proliferation and may cause fewer severe side effects.