Effects of RNA Interference ofTrypanosoma bruceiStructure-specific Endonuclease-I on Kinetoplast DNA Replication
Journal of Biological Chemistry
Kinetoplast DNA, the mitochondrial DNA of trypanosomatid protozoa, is a network containing several thousand topologically interlocked DNA minicircles. Kinetoplast DNA synthesis involves release of minicircles from the network, replication of the free minicircles, and reattachment of the progeny back onto the network. One enzyme involved in this process is structure-specific endonuclease-I. This enzyme, originally purified from Crithidia fasciculata, has been proposed to remove minicircle
... e minicircle replication primers (Engel, M. L., and Ray, D. S. (1998) Nucleic Acids Res. 26, 4773-4778). We have studied the structure-specific endonuclease-I homolog from Trypanosoma brucei, showing it to be localized in the antipodal sites flanking the kinetoplast DNA disk, as previously shown in C. fasciculata. RNA interference of structure-specific endonuclease-I caused persistence of a single ribonucleotide at the 5 end of both the leading strand and at least the first Okazaki fragment in network minicircles, demonstrating that this enzyme in fact functions in primer removal. Probably because of the persistence of primers, RNA interference also impeded the reattachment of newly replicated free minicircles to the network and caused a delay in kinetoplast DNA segregation. These effects ultimately led to shrinkage and loss of the kinetoplast DNA network and cessation of growth of the cell. . 2 The abbreviations used are: kDNA, kinetoplast DNA; SSE1, structure-specific endonuclease-I; TdT, terminal deoxynucleotidyl transferase; PBS, phosphate-buffered saline; GFP, green fluorescent protein; RNAi, RNA interference; dUTP, 2Ј-deoxyuridine 5Ј-triphosphate; dATP, 2Ј-deoxyadenosine 5Ј-triphosphate; DAPI, 4Ј,6-diamidino-2phenylindole.