Effects of RNA interference of Trypanosoma brucei structure-specific endonuclease-I on kinetoplast DNA replication.
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 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.
Liu
Yanan
Y
Department of Biological Chemistry, Johns Hopkins Medical School, Baltimore, Maryland 21205, USA.
Motyka
Shawn A
SA
Englund
Paul T
PT
D000818:Animals; D015152:Blotting, Northern; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018105:DNA, Kinetoplast / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000502:physiology*; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D034622:RNA Interference; D013997:Time Factors; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D018105:DNA, Kinetoplast; D049452:Green Fluorescent Proteins; D012313:RNA; D004247:DNA; D004720:Endonucleases; D004262:DNA Restriction Enzymes; C120663:structure-specific endonuclease I