Use of Divalent Metal Ions in the DNA Cleavage Reaction of Human Type II Topoisomerases

A number of essential nuclear processes, such as DNA recombination and replication, generate knots and tangles in the genetic material (1–3). DNA knots make it impossible to separate the two strands of the double helix, and tangled chromosomes cannot be segregated during mitosis or meiosis (2–5). Consequently, these detrimental DNA linkages must be resolved to maintain chromosomal integrity. The enzymes that remove knots and tangles from the genome are type II DNA topoisomerases (2, 4, 6–10). Type II topoisomerases act by passing an intact double helix through a transient double-stranded break that they generate in a separate segment of DNA (4, 7, 11–13). Humans encode two isoforms of the enzyme, topoisomerase IIα and IIβ (4, 7, 9, 10, 13). These isoforms differ in their protomer molecular masses (170 vs. 180 kDa, respectively) and are encoded by separate genes (2, 4, 9, 10). The physiology of topoisomerase IIα and IIβ differs greatly. Topoisomerase IIα is essential for the survival of proliferating cells (14). Enzyme levels are low in quiescent cells and increase dramatically during periods of growth (15–17). Furthermore, topoisomerase IIα is regulated over the cell cycle, with protein concentrations peaking in G2/M (17–19). In contrast, topoisomerase IIβ is dispensable at the cellular level, and its expression is independent of proliferative status and cell cycle (20, 21). Finally, while topoisomerase IIα is associated with replication forks and remains tightly bound to chromosomes during mitosis (6, 14, 22–24), the β isoform dissociates from chromosomes during mitosis (9, 22, 25). Despite the distinct expression patterns and cellular roles of the two isoforms, topoisomerase IIα and IIβ display a high degree (~70%) of amino acid sequence identity and often show similar enzymological characteristics (2, 9, 10). However, numerous studies of enzyme-mediated DNA cleavage and ligation suggest that there are significant (but subtle) differences in the active sites of topoisomerase IIα and IIβ. First, the DNA cleavage specificities of the two isoforms are similar, but not identical (26–28). Second, some anticancer drugs that enhance topoisomerase II-mediated DNA cleavage display preferential effects on one isoform or the other (28–30). Third, covalent topoisomerase IIα-cleaved DNA complexes (i.e., cleavage complexes) that are formed during scission generally persist longer than equivalent complexes with topoisomerase IIβ (29, 31). Fourth, topoisomerase IIβ is more sensitive than topoisomerase IIα to alterations in the scissile bond of DNA. For example, topoisomerase IIβ is less able to cleave a DNA substrate that contains a 3′-bridging sulfur atom (i.e., phosphorothiolate) in place of the normal oxygen atom (32). It also ligates a nick that has been activated by the addition of a 5′-pnitrophenyl group more slowly (33). All type II topoisomerases require a divalent metal ion in order to cleave and ligate DNA. Recently, it was shown that human topoisomerase IIα utilized a two-metal-ion mechanism similar to that used by primases, some DNA polymerases, and bacterial DNA gyrase (34–38). Given the differences in the active sites of topoisomerase IIα and IIβ, we wanted to determine whether the two isoforms used divalent metal ions in a similar manner. Results indicate that topoisomerase IIβ also utilizes a two-metal-ion mechanism for DNA cleavage. Furthermore, they provide the first kinetic evidence for an important interaction between a divalent metal ion and the non-bridging atom of the scissile phosphate in the DNA cleavage reaction of type II topoisomerases.