Intramitochondrial localization of universal minicircle sequence-binding protein, a trypanosomatid protein that binds kinetoplast minicircle replication origins

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithid ia fasciculata, is a unique structure containing 5,000 DNA minicircles topo logically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at uniqu e origins that are bound by universal minicircle sequence (UMS), binding pr otein (UMSBP), a sequence-specific DNA-binding protein. This protein, encod ed by a nuclear gene, localizes within the cell's single mitochondrion. Usi ng immunofluorescence, we found that UMSBP localizes exclusively to two nei ghboring sites adjacent to the face of the kDNA disk nearest the cell's fla gellum. This site is distinct from the two antipodal positions at the perim eter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-s tate levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesi s throughout the cell cycle, immunofluorescence indicated that UMSBP locali zation within the kinetoplast is not static. The intramitochondrial localiz ation of UMSBP and other kDNA replication enzymes significantly clarifies o ur understanding of the process of kDNA replication.