Topoisomerases: A Biochemical Overview
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Topoisomerase
Topoisomerases are a class of enzymes that create miniscule, reversible cuts in the DNA helix past the replication fork to relieve torsional stress, and stabilize the DNA helix during replication and transcription. The molecular structure of DNA is controlled by the aforementioned snipping of DNA and passing the strand through the cut. Type I topoisomerases create single stranded cuts in DNA, while Type II topoisomerases create double stranded cuts in DNA. Topoisomerases are further along the DNA helix past the replication fork, which contributes to it's ability to prevent breakage in DNA strands.
Function
During DNA replication and repair, chromosomes can become entangled. In order to prevent cytotoxic or mutagenic DNA strand breaks, Topoisomerase can disentangle DNA segments and add or remove supercoils. Several different types of Topoisomerase exist, which can be categorized into type I or type II, depending on whether they cut one or two strands of DNA. Both types contain the nucleophilic tyrosine, which is used to promote strand scission. [5]
Type I Mechanisms: -Type IA and II attach to 5’ ends of DNA, then pass a single stranded segment of DNA through a transient break in a second single strand of DNA
-Type IB and IC attach to 3’ ends of DNA, then nick one DNA strand, which allows one duplex end to rotate around the remaining phosphodiester bond
Type II Mechanism: Cleave both strands of a duplex DNA strand and pass another duplex DNA strand through the transient break
Disease
When not removed, supercoils within DNA can interfere with normal DNA replication. RNA and DNA hybrids, known as R-loops, can also form as a result of negatively supercoiled DNA. R-loops can also stall both replication and transcription processes and ultimately cause DNA double strand breaks. It follows then that binding of RNA processing factors to prevent RNA from forming R-loops is critical for normal cell function. While it is unlikely to be the only kinase to phosphorylate splicing factors, Topoisomerase 1 is known to promote spliceosome assembly through phosphorylation.
Several different malfunctions of TOP1 have been linked to mutagenesis or cell death. In one instance, TOP1 gets stuck on the DNA strand, due to the TOP1 and DNA cleavage complex (TOP1cc) being covalently linked and the topoisomerase reaction being aborted prematurely. Additionally, erroneous ribonucleotides within the sequence can cause permanent cuts to the strand by topoisomerase known as single strand breaks. Both types of errors can result in mutagenesis or cell death, which are precursors to tumorigenesis.
In yeast, mutations were found to arise from errors with TOP1cc. If similar errors were to be found in humans, it is expected that they would be linked to tumorigenesis. However, little research has been done that supports this hypothesis. Instead, recent studies have found that TOP1 is regulated quite differently within human cells.
Genetic mutations that directly impair the function of topoisomerases have correlation to autism spectrum disorders (ASDs) and similar neurological disorders. These genetic mutations cause the elongating of particular genes that are correlated to the expression of ASDs. Specifically, Ube3a is one of hundreds of genes that are associated with the development of ASDs in humans when elongated.
Outside of neurodevelopmental disorders, TOP1 disorders involving autoimmunity are common. Scleroderma, a group of diseases characterized by the production of antibodies that causes the hardening of connective tissues and skin, have been correlated with high levels of TOP1 antibodies produced within patients.
Relevance
Chemical or genetic interference of the mutated topoisomerases has been shown to have profound effects on function. Inhibitors of TOP1, such as topotecan, have been shown to decrease the expression of the elongated genes closely associated with ASDs. Specifically, this can be made true due to the fact that TOP1 is involved in the recruitment of spliceosomes to promote effective transcription, so TOP1 inhibitors directly effect spliceosome stabilization of R-loop formation, which inhibits the elongation and expression of the Ube3a gene.
Structural highlights
The primary structure of TOP1 can be divided into three regions. First, the N-terminus contains 214 amino acids, the core region contains 498 amino acids, and the C-terminus contains 53 amino acids. TOP1 consists of a multitude of various amino acids, but the active site consists of tyrosine residues in the C-terminus.
The secondary structure consists of right-handed alpha helices and antiparallel beta strands, which makes up beta sheets. The enzyme consists of 11 alpha helices and 12 beta strands. The clustering of beta sheets in this particular structure of TOP1 creates 3 beta sheets.
The tertiary structure consists of several motifs and domains. The motifs present are alpha bundles, alpha non-bundles, beta rolls, and beta ribbons. The domain consists of five residues of tyrosine and is a loop-like shape. This loop serves as the active site for the change in conformation, which allows for DNA helices entry.
The oligomeric state of the quaternary structure is heterotetrameric. There is no symmetry in this particular enzyme, due to the presence of various distinct subunits.
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References
1. Nichols, M.D., DeAngelis, K., Keck, J.L. and Berger, J.M. Structure and function of an archaeal topoisomerase VI subunit with homology to the meiotic recombination factor Spo11. The EMBO Journal [Online], (1999), 18: 6177-6188.
2. Min Li, Yilun Liu. Topoisomerase I in Human Disease Pathogenesis and Treatments. Genomics, Proteomics & Bioinformatics [Online], (2016), 14, 3: 166-171.
3. Vos, Seychelle M. Tretter, Elsa M. Schmidt, Bryan H. Berger, James M. All tangled up: how cells direct, manage and exploit topoisomerase function. Nature Reviews Molecular Cell Biology [Online], (2011), 12, 12: 827-841.
4. Takahashi DT, Gadelle D, Agama K, Kiselev E, Zhang H, Yab E, Petrella S, Forterre P, Pommier Y, Mayer C. Topoisomerase I (TOP1) dynamics: conformational transition from open to closed states. Nat Commun [Online], (2022), 13: 59.
5. Benjamin D. Bax, Garib Murshudov, Anthony Maxwell, Thomas Germe. DNA Topoisomerase Inhibitors: Trapping a DNA-Cleaving Machine in Motion. Journal of Molecular Biology [Online], (2019), 431, 18: 3427-3449.
6. Natassja G. Bush, Katherine Evans-Roberts, Anthony Maxwell. DNA Topoisomerases. EcoSal Plus [Online], (2015), Volume 6, Number 2, Domain: Synthesis and Processing of Macromolecules.
7. Diane T. Takahashi, Daniele Gadelle, Keli Agama, Evgeny Kiselev, Hongliang Zhang, Emilie Yab, Stephanie Petrella, Patrick Forterre, Yves Pommier, Claudine Mayer. Topoisomerase I (TOP1) dynamics: conformational transition from open to closed states. Nature Communications [Online] (2022), 13, 59.
