DNA topoisomerase is an enzyme found in all forms of life, recognized as a master regulator of DNA structure. Its general purpose is to manage the topological state of the DNA molecule, which is the way the strands are intertwined in three-dimensional space. Without the actions of topoisomerase, fundamental genomic processes such as DNA replication and transcription would rapidly halt. This enzyme resolves physical obstacles by temporarily altering the double helix, ensuring the genetic material remains accessible and untangled for cellular machinery.
The Structural Problem Topoisomerase Solves
The intertwined nature of the DNA double helix presents a challenge during cellular activities that require the strands to separate. When the two strands of the helix are unwound, the remaining double helix ahead of the unwinding point becomes excessively twisted. This forced overwinding creates a buildup of positive supercoiling, similar to twisting a rubber band until it coils upon itself.
This torsional stress must be relieved. If this tension is not resolved, the supercoils accumulate, causing the DNA or RNA polymerase enzymes involved in replication and transcription to stall. This effectively prevents the cell from dividing or expressing its genes.
Another problem the enzyme solves is the entanglement of DNA molecules, known as catenation, which happens when newly replicated daughter chromosomes remain linked. Topoisomerases are the sole enzymes capable of performing the transient breaking and rejoining required to pass one segment of DNA through another.
How Topoisomerase Recognizes DNA
The precise location where topoisomerase binds to DNA is determined by the geometry and tension of the helix rather than a specific sequence of nucleotides. Since they are non-sequence specific, they can act upon numerous sites across the genome where the DNA structure is strained. This allows them to act broadly wherever topological problems arise.
The two main types of the enzyme, Type I and Type II, show distinct binding preferences based on their function. Type I topoisomerases target regions under torsional strain, often requiring a single-stranded region to initiate binding. Type IA enzymes bind to single-stranded segments in negatively supercoiled DNA, while Type IB enzymes recognize double-stranded DNA under strain.
Type II topoisomerases bind to two separate double-stranded DNA segments, often recognizing a crossover point or segment geometry. The segment that will be cleaved is called the G-segment, and the segment that will be passed through the break is the T-segment. The enzyme recognizes the overall shape of the double helix to position itself for the subsequent cleavage reaction.
Cleavage site selection often depends more on the reactivity of a site under strain than the initial binding affinity. Analysis of eukaryotic Type IB cleavage sites suggests a slight preference against certain nucleotides, such as Guanine, near the cleavage point. This indicates that local sequence context can influence the efficiency of the enzyme’s action.
The Cleavage and Rejoining Mechanism
After initial binding to the strained DNA segment, the enzyme initiates its catalytic cycle by temporarily breaking the phosphodiester backbone of one or both strands. This break is achieved through a transesterification reaction, which conserves the energy of the cleaved bond. The enzyme forms a transient covalent intermediate, known as the cleavage complex.
This covalent attachment involves the nucleophilic attack of a hydroxyl group on an active site tyrosine residue onto a DNA phosphate group. The polarity of this linkage differs between the enzyme types: Type IB topoisomerases link to the 3′-phosphate end of the cleaved strand. In contrast, Type IA and Type II topoisomerases attach to the 5′-phosphate end.
The transient break allows for the topological change to occur through a strand passage mechanism. Type I topoisomerases permit the untethered strand to rotate around the intact strand, thereby relaxing the supercoiling. Type II topoisomerases form a double-strand break in the G-segment and use jaw-like structures to pass the entire T-segment double helix through the gap.
Once the topological problem is resolved, the enzyme completes the cycle by reversing the transesterification reaction, rejoining the DNA backbone. The 3′-hydroxyl group of the cleaved strand performs a second nucleophilic attack on the phosphotyrosyl bond, displacing the enzyme and restoring the original phosphodiester bond. This re-ligation step releases the enzyme.
Topoisomerase as a Target in Medicine
The mechanism of topoisomerase cleavage and rejoining is exploited in medical treatments, particularly in cancer chemotherapy. Drugs known as topoisomerase poisons function by interfering with the enzyme’s cycle after the DNA cleavage step. These compounds stabilize the transient covalent topoisomerase-DNA cleavage complex.
By stabilizing this complex, the drugs prevent the enzyme from completing the necessary re-ligation of the broken DNA strands. The enzyme remains covalently bound to the broken DNA, causing unrepaired single or double-strand breaks in the genome.
The resulting DNA damage triggers programmed cell death, or apoptosis, in rapidly dividing cells. Specific drugs are designed to target either Type I or Type II enzymes, often by intercalating at the interface between the cleaved DNA ends and the enzyme to physically block the re-ligation process.