Abstract
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.
Highlights
The T. brucei kinetoplast DNA (kDNA) network contains several thousand minicircles that are heterogeneous in sequence and a few dozen maxicircles
The predicted T. brucei SSE1 protein (295 amino acids) is 63% identical to its C. fasciculata counterpart (297 amino acids), and there are no other genes closely related to SSE1 in the T. brucei genome
The T. brucei SSE1 is concentrated in the antipodal sites flanking the kDNA disk (Fig. 1A), the same location previously reported for the C. fasciculata enzyme [16]
Summary
The T. brucei kDNA network contains several thousand minicircles that are heterogeneous in sequence (each 1 kb) and a few dozen maxicircles (each 23 kb). The progeny minicircles, containing nicks or gaps, migrate to two antipodal sites that flank the kDNA disk [11] Within these sites, subsequent steps of replication occur, catalyzed by enzymes localized therein. Subsequent steps of replication occur, catalyzed by enzymes localized therein These reactions are thought to include primer removal by a structure-specific endonuclease-I (SSE1) [15,16,17] and repair of some (but not all) of the minicircle gaps by a DNA polymerase  [18, 19] and a DNA ligase [20, 21]. We found that SSE1 RNAi affects segregation of the kDNA network after its replication
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