Topoisomerases and cancer chemotherapy: recent advances and unanswered questions

Abstract

DNA topoisomerases are enzymes that catalyze changes in the torsional and flexural strain of DNA molecules. Earlier studies implicated these enzymes in a variety of processes in both prokaryotes and eukaryotes, including DNA replication, transcription, recombination, and chromosome segregation. Studies performed over the past 3 years have provided new insight into the roles of various topoisomerases in maintaining eukaryotic chromosome structure and facilitating the decatenation of daughter chromosomes at cell division. In addition, recent studies have demonstrated that the incorporation of ribonucleotides into DNA results in trapping of topoisomerase I (TOP1)-DNA covalent complexes during aborted ribonucleotide removal. Importantly, such trapped TOP1-DNA covalent complexes, formed either during ribonucleotide removal or as a consequence of drug action, activate several repair processes, including processes involving the recently described nuclear proteases SPARTAN and GCNA-1. A variety of new TOP1 inhibitors and formulations, including antibody-drug conjugates and PEGylated complexes, exert their anticancer effects by also trapping these TOP1-DNA covalent complexes. Here we review recent developments and identify further questions raised by these new findings.

Keywords: DNA supercoiling; DNA-activated protease; DNA-protein crosslink; chromatin organization; topoisomerase poison.

Figure 1.. Topoisomerase mechanisms.

In the topoisomerase I cleavage complex (TOP1cc) (top), the 3’ DNA end is covalently linked to the active site tyrosine (Y). Changes in the linkage of DNA strands occur through a mechanism of strand rotation, where the untethered 5’ DNA end of the cleaved strand swivels about the noncleaved DNA strand. TOP2 (middle) and TOP3 (bottom) both involve mechanisms of DNA strand transfer. In the case of TOP2cc, the G segment of duplex DNA is cleaved by the two active sites of the homodimer, following capture of the T segment by the closure of the N-terminal ATPase domains. The T segment DNA is then successively passed through the double-strand break in the G segment and out through the bottom dimer interface. For type IA enzymes, depicted for TOP3cc, a single strand of negatively supercoiled DNA is cleaved to form a 5’ phosphotyrosyl bond, while the 3’OH end is held by the enzyme. A conformational change in the protein then allows the intact complementary strand to be passed through the protein-linked break, followed by religation of the cleaved DNA.

Figure 2.. Distinct actions of topoisomerase poisons and inhibitors.

( A) As diagrammed for TOP1, a canonical inhibitor would prevent enzyme-mediated cleavage of a single strand of duplex DNA, while a poison (such as camptothecin) acts to stabilize the topoisomerase I cleavage complex (TOP1cc) reaction intermediate, thereby converting a normal enzyme into a source of DNA damage. The same principles apply to TOP2, although, in these instances, the dimeric enzymes produce two enzyme-linked DNA breaks staggered by 4 bp. ( B) Based on these distinct modes of action, increased topoisomerase levels in an isogenic cell line would induce opposing effects on drug sensitivity: resistance to an inhibitor versus increased sensitivity to a poison. Shown in this diagram are the dose response curves for killing that result from an increase in topoisomerase levels relative to cells that yield the black curve.