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Analysis of the Repair of
Topoisomerase II DNA Damage

By: Eric Goldstein | Mentor: Dr. Mark Muller


Topoisomerase poisons are widely used as anti-cancer chemotherapeutics [2]. Topoisomerase IIα (topoIIα) is the target for many anti-cancer agents because cancer cells have increased mitotic activity, requiring an increase in topoIIα expression.

Topoisomerase IIa Mechanism

TopoIIα stimulates relaxation, decatanation, and unwinding DNA during replication and cellular division (Figure 1). A prime example of topoIIα catalytic activity is during DNA replication when the replication fork melts the DNA hydrogen bonds between base pairs. As a result, the DNA preceding the replication fork begins to wind into a highly taut coil called a supercoil. If left alone, this negative supercoiling can be so severe as to fracture the DNA itself, thereby creating a genotoxic event [2]. TopoIIα relaxes the supercoiling.

TopoIIα is homodimer with a Mg2+ cation per dimer. TopoIIα exists as closed or open clamps dependent upon ATP binding. ATP binding switches topoIIα from an open to closed clamp formation. In each subunit of human topoIIα, the Mg2+ cation stabilizes the tyrosine – 804 residue, thus allowing a nucleophillic attack of the 5' phosphodiester bond [2]. The mechanism is repeated on both sides of the double helix. As a result, TopoIIα becomes covalently bound and creates a protein-DNA adduct with a double stranded break (DSB). Transient strand passage translocates the uncut strand through the DSB. Within the active site, the dissociated ends are religated. ATP hydrolysis then switches the homodimer to the open conformation. This mechanism is equilibrated and can either increase or decrease the linking number by two, meaning that both phosphodiester backbones are translocated, tightening or relaxing the helical structure. The isozyme topoIIβ is not mitotically stimulated and is poorly understood, but it is known to share this mechanism [2] (Figure 1).

Topoisomerase I Mechanism

Topoisomerase I (topoI) is relevant to mitotic, transcription, and promoter regulation [2]. TopoI does not require ATPase activity. A tyrosine residue performs a nucleophilic attack on the 5' phosphodiester bond, creating a single stranded gap. TopoI transfers the free 3' end about the intact strand and religates the gap within the catalytic site. The topoI mechanism is in equilibrium, allowing for the increase or decrease in linking number by one through the pivoting of one phosphodiester backbone around the other.

Figure 1 - A. Topoisomerase II Poison; B. Topoisomerase II Enzyme Mechanism [2]

Topoisomerase IIα Poisons

TopoII poisons such as etoposide (VP16) stabilize enzyme/DNA cleavages and fragment the genome (Figure 1). Many topoIIα agents are in clinical use and are approved by the Food and Drug Administration [5]. Therefore, it is vital that an understanding of how topoIIα breaks are repaired allowing cancer cells to elude treatment is necessary. VP16 itself is a widely used chemotherapeutic agent and readily available, thus will be the focus of this project [5].

Chemotherapy sometimes requires high dosages of topoIIα agent to ensure that DNA damage does not undergo repair, as the cleavage complex is a transient and reversible event [2]. The stabilized DSB created by topoIIα poisons increase the half-life of the cleavage complex. DNA/topoII complexes are processed by the 26S proteosome, a macromolecular structure that degrades ubiquitinated proteins, thus removing the topoIIα polypeptide portion and leaving a DSB (Figure 2) [4]. Recent studies indicate that the removal of the topoIIα protein can be performed through CtIP and the phosphodiesterases TDP1 and TDP2 [9]. If the DNA damage is not efficiently repaired, the cell will undergo apoptosis. This could possibly reduce the amount of agent needed to fight the malignancy. Information on the repair process can lead to new strategies that can inhibit the reversal of topoIIα mediated DNA damage, thereby minimizing patient side effects through the increase of drug efficacy.

Figure 2 - TopoII Cleavage Complex Repair [4]

DSB Repair Pathways

DSBs are common events. The dissociated ends created by DSBs can reassociate indiscriminately, differing in sequence from the wild type and thereby creating chromosomal translocations [7]. To circumvent this, cells evolved two known mechanisms to correct the DSB: non-homologous end joining (NHEJ) and homologous recombination (HR).

NHEJ is the main pathway by which healthy cells repair DSBs; however, this can alter gene regulation or expression (Figure 3). The process involves the direct ligation of a DSB without regard to sequence homology or phase of the cell cycle [12]. NHEJ is a low fidelity, high mutation prone pathway, but repairs DSBs rapidly [12]. Ku, a heterodimer of Ku70 and Ku80, recognizes the DSB and initiates the NHEJ repair pathway [12]. The Ku protein attracts DNA-PKcs by forming a holoenzyme and autophosphorylates itself, possibly providing the energy needed for the subsequent blunt ligation. NHEJ provides genomic stability with a half life of 30 minutes [12].

HR is a high fidelity but time-consuming pathway occurring mainly in the late phases of the cell cycle [1]. HR commences upon DNA damage recognition, and a cascade of signaling recruits proteins that further resect the break to single stranded 3' ends (Figure 3) [8]. The single stranded ends are then coated with single stranded binding proteins, protecting the templates. These unbound ends are then wrapped with Rad51, which is associated with BRCA1. With Rad51 bound, the single stranded DNA participates in homology recognition [1]. The Rad51 complex also allows for strand exchange. Subsequent branch migration and nucleotide polymerization from DNA polymerase II occur. The whole complex then resolves itself with an exact copy of the template homolog where the DSB occurred. Most non-cancer cells are in the resting phases of the cell cycle and thus are not subjected to the HR pathway often.


This research project analyzes whether topoII/DNA damage complexes are repaired through either HR or NHEJ. DNA repair events resulting from poison damage can be quantified through the use of a highly specific reporter cassette for either HR or NHEJ. With this experimental system, we found that HR is the preferred DSB repair pathway in HeLa cells. This knowledge could lead to increased efficacy of anti-cancer chemotherapeutics by blocking HR pathway proteins and/or signaling.

Figure 3 - NHEJ [11] and HR [7] Pathways. NHEJ uses the Ku complex to recruit subsequent proteins such as DNA-PKcs, ultimately resulting in blunt double strand ligation of the DSB. HR is a high fidelity pathway that uses a donor sequence as a template, thus resulting in high fidelity DNA retrieval.

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