The kinetoplast structure-specific endonuclease I is related to the 5' exo/endonuclease domain of bacterial DNA polymerase I and colocalizes with the kinetoplast topoisomerase II and DNA polymerase beta during replication.
The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata has an unusual structure composed of minicircles and maxicircles topologically interlocked into a single network and organized in a disc-shaped structure at the base of the flagellum. We previously purified a structure-specific endonuclease (SSE1), based on its RNase H activity, that is enriched in isolated kinetoplasts. The endonuclease gene has now been cloned, sequenced, and found to be closely related to the 5' exonuclease domain of bacterial DNA polymerase I proteins. Although the protein does not contain a typical mitochondrial leader sequence, the enzyme is shown to colocalize with a type II DNA topoisomerase and a DNA polymerase beta at antipodal sites flanking the kinetoplast disc. Cell synchronization studies with an epitope-tagged construct show that the localization of the endonuclease to the antipodal sites varies in a cell cycle-dependent manner similar to that of the DNA polymerase beta [Johnson, C. E. & Englund, P. T. (1998) J. Cell Biol. 143, 911-919]. Immunofluorescent localization of SSE1 to the antipodal sites is only observed during kinetoplast replication. Together, these results suggest a point of control for kinetoplast DNA replication through the regulation of the availability of DNA replication proteins and a possible role for the antipodal sites in removal of RNA primers and the repair of gaps in newly replicated minicircles.
Molecular Biology Institute, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095-1570, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D003001:Cloning, Molecular; D003421:Crithidia / Q000201:enzymology*; D004256:DNA Polymerase I / Q000737:chemistry*; D019951:DNA Polymerase beta / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics; D007211:Indoles; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D016914:Ribonuclease H / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D004272:DNA, Mitochondrial; D007211:Indoles; D011994:Recombinant Proteins; C007293:DAPI; D004256:DNA Polymerase I; D019951:DNA Polymerase beta; D016914:Ribonuclease H; D004250:DNA Topoisomerases, Type II