A structure-specific DNA endonuclease is enriched in kinetoplasts purified from Crithidia fasciculata

Abstract

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.

Effects of RNA Interference of Trypanosoma brucei Structure-specific Endonuclease-I on Kinetoplast DNA Replication

“…The subject of this paper is the SSE1, a mitochondrial enzyme discovered in C. fasciculata by Engel and Ray (15)(16)(17). As mentioned above, this enzyme localizes to the antipodal sites.…”

“…Effect of RNAi on Replication Primers-The enzymatic properties of purified SSE1 had implied a function in primer removal (15,17). To test this possibility in vivo, we compared the size of minicircle primers in uninduced cells with those in cells induced for RNAi for 2 or 4 days.…”

“…As mentioned above, this enzyme localizes to the antipodal sites. Enzymatic studies on purified C. fasciculata SSE1 protein showed that it has RNase H and DNA endonuclease activities (15) similar to those of the FEN family of enzymes (30,31). It is homologous to the 5Ј exonuclease domain of Escherichia coli DNA polymerase I (16).…”

“…Within these sites, subsequent steps of replication occur, catalyzed by enzymes specifically 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). Finally, the progeny minicircles, still containing at least one nick or gap, are attached to the edge of the kDNA network by an antipodal topoisomerase II (22,23).…”

A Second Mitochondrial DNA Primase Is Essential for Cell Growth and Kinetoplast Minicircle DNA Replication in Trypanosoma brucei

“…Minicircle replication intermediates and other replication proteins were subsequently localized to the antipodal sites (12,18). These include a ␤-type DNA polymerase (38), a structure-specific endonuclease (SSE1) (8,10), a DNA ligase (ligase k␤) (42), RNase H (9), two DNA helicases (PIF1 and PIF5) (28,29), a H1 histone-like protein (KAP4) (49), a minicircle origin binding protein (p38) (26), a type IA topoisomerase (39), and a DNA primase (PRI1) (16). At least partial repair of discontinuities in nascent minicircles is proposed to occur at these sites (15).…”