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

Abstract

. These primases are members of the archeoeukaryotic primase superfamily, and each of them contain an RNA recognition motif and a PriCT-2 motif. In Leishmania species, PRI2 proteins are approximately 61 to 66 kDa in size, whereas in Trypanosoma species, PRI2 proteins have additional long amino-terminal extensions. RNA interference (RNAi) of T. brucei PRI2 resulted in the loss of kinetoplast DNA and accumulation of covalently closed free minicircles. Recombinant PRI2 lacking this extension (PRI2⌬NT) primes poly(dA) synthesis on a poly(dT) template in an ATP-dependent manner. Mutation of two conserved aspartate residues (PRI2⌬NTCS) resulted in loss of enzymatic activity but not loss of DNA binding. We propose that PRI2 is directly involved in initiating kinetoplast minicircle replication.The mitochondrial DNA (kinetoplast DNA [kDNA]) of trypanosomes has long been known to have a novel network structure consisting of topologically interlocked minicircles and maxicircles (30). The kDNA exists as a highly condensed DNA-protein disc located close to the base of the flagellum, with the kDNA connected to the basal body through a transmembrane filament system termed the tripartite attachment complex (32). Each cell contains a single kDNA network that must be duplicated, with the daughter networks segregating into each of the two daughter cells. Networks contain several thousand minicircles and only 20 to 30 maxicircles. In Trypanosoma brucei, the maxicircles are 23 kb in size, and the minicircles are 1 kb. The maxicircles encode genes for mitochondrial ribosomal RNAs and genes encoding proteins involved in oxidative metabolism. Minicircles encode small RNA species termed guide RNAs, which serve as templates in the insertion or deletion of U residues in the maxicircle transcripts by a mechanism termed RNA editing (44).Early studies of this unusual form of mitochondrial DNA focused on the mechanisms of replication of the DNA using light and electron microscopies, radioactive labeling, sedimentation, gel electrophoresis, and restriction enzyme analysis. Detailed studies of minicircle replication were made possible by the finding which showed that minicircles are released one at a time from the center of the kDNA network and replicate free of the network (11). The newly replicated minicircles are then rejoined to the periphery of the network, and the network eventually doubles in size and is divided by unknown mechanisms to yield two daughter networks (22,25).Minicircles replicate by a unidirectional theta-type intermediate from an origin sequence that is specified by the universal minicircle sequence (UMS) (1,2,33,34,46). One strand (leading strand) is synthesized continuously, and the other strand (lagging strand) is synthesized discontinuously (3,20,21,37,41). The leading strand is initiated within the UMS by an RNA priming mechanism and is elongated past a nearby hexamer sequence where the first Okazaki fragment is initiated. A DNA-binding protein (UMSBP) binds both to the UMS and to the hexamer sequence in their single-...

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

“…The majority of kDNA replication proteins studied localize to specific regions surrounding the kDNA, mainly the antipodal sites and the KFZ (17,18,28,29,45). Previously, we demonstrated that TbPOLID is one of three mitochondrial DNA polymerases that are essential for parasite survival and the maintenance of the kDNA network (6).…”

“…RNA editing depends upon guide RNAs (gRNAs) encoded on the heterogeneous population of 5,000 minicircles (1 kb) (49). Therefore, the coordinated replication of both minicircles and maxicircles is essential for mitochondrial physiology and parasite survival.Key features of the kDNA replication mechanism include replication once per cell cycle in near synchrony with nuclear S phase, a topoisomerase II-mediated release and reattachment of minicircles, and a multiplicity of DNA polymerases (six), helicases (six), and primases (two) for kDNA transactions (17,18,22,27,45). Briefly, covalently closed minicircles are released from the network into the kinetoflagellar zone (KFZ), a specialized region between the kDNA disk and the mitochondrial membrane nearest the basal body (bb) (10).…”

A Passion for Parasites

“…We never identified the gene for this enzyme. Jane Hines and Dan Ray (Jane is Dan's wife) subsequently identified genes for two additional mitochondrial primases (57,58), and neither one appeared to be Congjun's enzyme.…”

Three Mitochondrial DNA Polymerases Are Essential for Kinetoplast DNA Replication and Survival of Bloodstream Form Trypanosoma brucei

“…A plethora of proteins involved in kDNA replication have been studied in T. brucei, including six helicases (25)(26)(27)40), two DNA ligases (10), two primases (19,20), a topoisomerase IA (40), a topoisomerase II (50), and five DNA polymerases (Pols) (4,7,21,35). The involvement of multiple DNA polymerases in kDNA replication distinguishes this process from replication of other mitochondrial genomes, which depend solely upon DNA polymerase ␥, a family A DNA polymerase.…”

“…POLIB, POLIC, and POLID lack homologues in mammals, including humans, thus identifying these proteins as potential biological targets for the development of new antitrypanosomal drugs. Analyses of kDNA replication proteins have provided compelling molecular evidence for essential functions in distinct steps of kDNA replication in procyclic form (PF) parasites, a life cycle stage found in its insect vector (4,7,20,26). However, analysis of kDNA replication protein functions in bloodstream form (BF) parasites, the life cycle stage found in the mammalian host and the target for disease intervention (18,37), is an understudied area of trypanosome biology.…”