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NKX3.1 Homeodomain Protein Binds to Topoisomerase I and Enhances Its Activity

¹ Departments of Oncology and Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, District of Columbia and ² Laboratory of Proteomics and Analytical Technologies, Science Applications International Corporation-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland

Requests for reprints: Edward P. Gelmann, Departments of Oncology and Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, 3800 Reservoir Road Northwest, Washington, DC 20007-2197. Phone: 202-444-2207; Fax: 202-444-1229; E-mail: gelmanne{at}georgetown.edu.

The prostate-specific homeodomain protein NKX3.1 is a tumor suppressor that is commonly down-regulated in human prostate cancer. Using an NKX3.1 affinity column, we isolated topoisomerase I (Topo I) from a PC-3 prostate cancer cell extract. Topo I is a class 1B DNA-resolving enzyme that is ubiquitously expressed in higher organisms and many prokaryotes. NKX3.1 interacts with Topo I to enhance formation of the Topo I-DNA complex and to increase Topo I cleavage of DNA. The two proteins interacted in affinity pull-down experiments in the presence of either DNase or RNase. The NKX3.1 homeodomain was essential, but not sufficient, for the interaction with Topo I. NKX3.1 binding to Topo I occurred independently of the Topo I NH₂-terminal domain. The binding of equimolar amounts of Topo I to NKX3.1 caused displacement of NKX3.1 from its cognate DNA recognition sequence. Topo I activity in prostates of Nkx3.1^+/– and Nkx3.1^–/– mice was reduced compared with wild-type mice, whereas Topo I activity in livers, where no NKX3.1 is expressed, was independent of Nkx3.1 genotype. Endogenous Topo I and NKX3.1 could be coimmunoprecipitated from LNCaP cells, where NKX3.1 and Topo I were found to colocalize in the nucleus and comigrate within the nucleus in response to either -irradiation or mitomycin C exposure, two DNA-damaging agents. This is the first report that a homeodomain protein can modify the activity of Topo I and may have implications for organ-specific DNA replication, transcription, or DNA repair. [Cancer Res 2007;67(2):455–64]

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