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© The Rockefeller University Press, 0021-9525/2001//725 $5.00
The Journal of Cell Biology, Volume 153, Number 4, , 2001 725-734

Intramitochondrial Localization of Universal Minicircle Sequence-Binding Protein, a Trypanosomatid Protein That Binds Kinetoplast Minicircle Replication Origins

^a Department of Parasitology, The Kuvin Center for the Study of Infectious and Tropical Diseases, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
^b Department of Biological Chemistry, Johns Hopkins Medical School, Baltimore, Maryland 21205
Dept. of Parasitology, The Kuvin Center for the Study of Infectious and Tropical Diseases, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.972-2-6757425972-2-6758089

Shlomai{at}cc.huji.ac.il

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase β, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication.

Key Words: kinetoplast DNA • kDNA replication • universal minicircle sequence • UMS-binding protein

© 2001 The Rockefeller University Press

Abbreviations used in this paper: CaBP, calcium-binding protein; kDNA, kinetoplast DNA; pol, DNA polymerase; SSE1, structure-specific endonuclease I; TdT, terminal deoxynucleotidyl transferase; UMS, universal minicircle sequence; UMSBP, UMS-binding protein.

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