Human DNA Topoisomerase IIα Research Articles

Homologous and Heterologous Protein–Protein Interactions of Human DNA Topoisomerase IIα

DNA topoisomerase II (topo II; EC 5.99.1.3) is a nuclear enzyme whose DNA decatenating activity on newly replicated DNA is essential to successful cell division. Topo II catalytic activity proceeds by a concerted DNA breakage–reunion reaction coordinated between two interacting, homologous subunits. Human and yeast topo II have recently been shown to enter into heterologous protein–protein interactions and some of these interactions appear necessary for successful chromosomal segregation. In the present study, the sequences mediating homologous and heterologous protein–protein interactions have been investigated biochemically using various truncated peptides from the major α form of human topo II. From nonreducing gel electrophoresis and solid-phase protein–protein binding (Far Western) assays, topo II homodimerization appeared to be minimally governed by the region between amino acids 951 and 1042. However, maximal homodimerization and multimerization required sequences C-terminal to position 1042. Topo II peptides were also able to interact with 10–12 nuclear proteins from HeLa cells, termed topo II-interactive proteins or TIPs. Interestingly, small topo II peptides between residues 808 and 951 that did not homodimerize with topo II (857–1447) were nonetheless capable of binding to HeLa TIPs. These interactions were confirmed by use of topo II affinity chromatography for isolation of specific TIPs from HeLa nuclear extracts. Taken together, these data confirm that human topo II is also capable of heterologous interactions with nuclear proteins and that the region governing these interactions is distinct from, but has some overlap with, sequences directing topo II homodimerization.

NF-M trans-activates the human DNA topoisomerase IIα promoter independently of c-Myb in HL-60 cells

Identifying transcriptional regulators of DNA topoisomerase IIα (topo IIα) is essential to decipher the mechanisms underlying leukemia cell resistance to topo II-directed antitumor drugs. We have previously reported that the proto-oncogene transcription factor c-Myb trans activates the topo IIα promoter in several hematopoietic cell lines. Currently, we investigate whether NF-M, a C/EBPβ family member, cooperates with c-Myb in activating topo IIα transcription. Although NF-M is the most efficacious trans-activator of topo IIα that we have examined (~38-fold over basal), NF-M does not appear to be involved in the endogenous transcriptional regulation of topo IIα. Interestingly, we report that the sodium butyrate-dependent induction of the topo IIα promoter observed previously appears to be mediated by c-Myb, independent of NF-M.