NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity.
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 gamma-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.
Bowen
Cai
C
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, District of Columbia 20007-2197, USA.
Stuart
August
A
Ju
Jeong-Ho
JH
Tuan
Jenny
J
Blonder
Josip
J
Conrads
Thomas P
TP
Veenstra
Timothy D
TD
Gelmann
Edward P
EP
D000818:Animals; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000378:metabolism; D004789:Enzyme Activation; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000378:metabolism*; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism*
D004273:DNA, Neoplasm; D018398:Homeodomain Proteins; C491178:NKX3-1 protein, human; D014157:Transcription Factors; D004264:DNA Topoisomerases, Type I