Topoisomerases: A Biochemical Overview

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 its ability to prevent breakage in DNA strands [1].

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 amino acid side chain tyrosine, which is used as a catalyst to promote strand scission [2].

Type I topoisomerase mechanisms consist of two subcategories. Type IA 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. Conversely, 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 topoisomerase have one mechanism wherein they cleave both strands of a duplex DNA strand and pass another duplex DNA strand through the transient break [2].

Type 1B topoisomerase enzymes are found in eukaryotes, including humans, and are commonly referred to as TOP1. The mechanism of action for TOP1 involves three domains of the enzyme (the capping module or CAP, the catalytic module or CAT, and the linker domain) which make up the core region medial to the N-terminus and C-terminus [3]. This portion of the protein folds around the DNA strand in order to operate on it and remove torsional stress [3]. See the structural highlights section below for further discussion on the structure of TOP1.

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, TOP1 is known to promote spliceosome assembly through phosphorylation [4].

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 (known as 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 [4].

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. At transcriptionally-active regions (known as TARs) human cells were found to actually suppress TOP1 activity through SUMO (small ubiquitin-like modifier, a family of proteins which can be covalently attached to other proteins to modify their function) modifications at lysine residues K391 and K436. These residues are found at the catalytic core of the TOP1 enzyme in mammal cells, but not in yeast cells. This suppression leads to less TOP1 induced DNA damage, and is believed to be a mechanism of preventing genome instability. However, as of now a definite link between defects in these residues and genome instability leading to mutagenesis and/or cancer are yet to be firmly established [4].

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 [4].

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 [4].

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 [5].

TOP1cc accumulation on DNA also plays a role in how some tumor treatments function. For instance, Camptothecin (CPT), an herbal compound used in traditional Chinese medicine for thousands of years as tumor treatment, was identified as targeting TOP1 in the 1980s. Since then, synthetic analogs of CPT called irinotecan and topotecan have been developed and used as chemotherapeutic drugs for particularly aggressive forms of cancer. CPT and its analogs act as TOP1 poisons, which have a high affinity for the DNA-TOP1 cleavage complex [5]. Camptothecin is known as an “interfacial inhibitor” due to its selective binding to the TOP1-DNA cleavage complex. As the name implies, this category of inhibitors interact with interface(s) found on macromolecules, in this example a specific binding site at the seam between TOP1 and DNA [6].

By binding to this complex and interfering with the topoisomerase reaction, these TOP1 poisons adhere the intermediate together covalently. This causes DNA damage and induces cell death, which fast-growing cancer cells are vulnerable to. It should be noted that while this treatment option is useful for particularly aggressive cancers, it can also induce a myriad of life-threatening side effects. Thus, it is used sparingly and requires a great deal more research and modification before it can be used as a widespread treatment for cancer [5].

Using YM155 (Sepantronium bromide) as an inhibitor for TOP1 and TOP2 can cause DNA damage checkpoint signalling which can be used during cancer treatment. This suppresses survivin, which is able to resist chemotherapy treatment. The inhibition of TOP1 and TOP2 with this method stops mitosis at the S and M phase. YM155 does this by competing with DNA binding sites on topoisomerase, making it non-poisonous to topoisomerase [7].

Structural highlights

The primary structure of TOP1 can be divided into three regions. First, the (highlighted in red) contains 214 amino acids, the (highlighted in green) contains 498 amino acids, and the (highlighted in purple) contains 114 amino acid residues [8].

The consists of right-handed alpha helices (highlighted in light blue) 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 (highlighted in green) [8].

The oligomeric state of the quaternary structure is . There is no symmetry in this particular enzyme, due to the presence of various distinct subunits [9].

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 (highlighted in purple) for the change in conformation, which allows for DNA helices entry [10].

Topoisomerases' structure has hinges that allow it to open and close so it can clamp onto DNA to bind with it. Additionally, it has cavities within it that allow it store DNA segments if needed. Topoisomerase also has a coupling of conformational changes to allow it to make changes in DNA movement and rotation [10].