A structure-specific DNA endonuclease is enriched in kinetoplasts purified from Crithidia fasciculata.
The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata consists of minicircles and maxicircles topologically interlocked in a single network per cell. Individual minicircles replicate unidirectionally from either of two replication origins located 180 degrees apart on the minicircle DNA. Initiation of minicircle leading-strand synthesis involves the synthesis of an RNA primer which is removed in the last stage of replication. We report here the purification to near homogeneity of a structure-specific DNA endo-nuclease based on the RNase H activity of the enzyme on a poly(rA).poly(dT) substrate. RNase H activity gel analysis of whole cell and kinetoplast extracts shows that the enzyme is enriched in kinetoplast fractions. The DNA endonuclease activity of the enzyme is specific for DNA primers annealed to a template strand and requires an unannealed 5' tail. The enzyme cleaves 3' of the first base paired nucleotide releasing the intact tail. The purified enzyme migrates as a 32 kDa protein on SDS gels and has a Stoke's radius of 21.5 A and a sedimentation coefficient of 3.7 s, indicating that the protein is a monomer in solution with a native molecular mass of 32.4 kDa. These results suggest that the enzyme may be involved in RNA primer removal during minicircle replication.
Engel
M L
ML
Molecular Biology Institute and Department of Molecular, Cell and Developmental Biology, 611 Circle Drive East, University of California Los Angeles, Los Angeles, CA 90095-1570, USA.
Ray
D S
DS
D000818:Animals; D020029:Base Pairing; D002458:Cell Fractionation; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D016831:Crithidia fasciculata / Q000201:enzymology* / Q000235:genetics; D017931:DNA Primers / Q000378:metabolism*; D018105:DNA, Kinetoplast / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D012313:RNA / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D018105:DNA, Kinetoplast; C034477:RNA primers; D012313:RNA; D003850:Deoxyribonuclease I; D016914:Ribonuclease H