Stem-Loop Silencing Reveals that a Third Mitochondrial DNA Polymerase, POLID, Is Required for Kinetoplast DNA Replication in Trypanosomes

Abstract

. Previously, we identified four family A DNA Pols from Trypanosoma brucei with similarity to bacterial DNA Pol I and demonstrated that two (POLIB and POLIC) were essential for maintaining the kDNA network, while POLIA was not. Here, we used RNA interference to investigate the function of POLID in procyclic T. brucei. Stem-loop silencing of POLID resulted in growth arrest and the progressive loss of the kDNA network. Additional defects in kDNA replication included a rapid decline in minicircle and maxicircle abundance and a transient accumulation of minicircle replication intermediates before loss of the kDNA network. These results demonstrate that POLID is a third essential DNA Pol required for kDNA replication. While other eukaryotes utilize a single DNA Pol (Pol ␥) for replication of mitochondrial DNA, T. brucei requires at least three to maintain the complex kDNA network.Trypanosoma brucei and related trypanosomatid parasites (T. cruzi and Leishmania spp.) cause fatal and disfiguring diseases and, subsequently, significant medical and economic stress worldwide, with nearly 500 million people at risk for these vector-borne diseases (7). Current drug treatments are toxic, and no vaccines are available (45). Trypanosomatids are also divergent eukaryotes with a number of unusual biological properties, but one of their most interesting features is their mitochondrial DNA, known as kinetoplast DNA (kDNA). Unlike any DNA structure in nature, kDNA is a network containing thousands of catenated circular DNA molecules (minicircles and maxicircles). Several dozen maxicircles (23 kb) and ϳ5,000 minicircles (1 kb) are condensed into a disk-shaped structure in a specialized region of the cell's single mitochondrion, which is linked to the flagellar basal body through a tripartite attachment complex (16,36,39).The kDNA network is essential for the survival of both procyclic and bloodstream forms of the parasite (42); therefore, understanding kDNA replication and repair processes is an important aspect of trypanosome biology. Network replication is complex, requiring coordinated duplication of each minicircle and maxicircle in near synchrony with nuclear DNA replication (during S phase) (50). Currently, trypanosomatids are the only known eukaryotes to contain at least six mitochondrial DNA polymerases (Pols), namely, two Pol ␤-type enzymes (typically a nuclear repair protein) and four family A Pols related to bacterial DNA Pol I (21, 40). This is in striking contrast to what is the case for other eukaryotes, which contain just one mitochondrial DNA Pol, Pol ␥, for replication and repair transactions.To overcome the topological constraints within the catenated network, a key feature of the replication mechanism is the topoisomerase II-mediated release of individual covalently closed (CC) minicircles into a specialized region called the kinetoflagellar zone (KFZ) (10). Here, the free minicircles initiate unidirectional theta structure replication. Several proteins considered to be involved in this process are also found in th...

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

“…Although our previous data on silencing TbPOLID revealed a role in minicircle replication, the maxicircle copy number declined more rapidly and completely than that of minicircles. These data suggested that POLID also has an important role in maxicircle replication (6). So far, only two proteins have definitive roles in maxicircle replication, the primase PRI1 and the helicase PIF2 (17,27).…”

“…Two distinct regions, the antipodal sites and the KFZ, have emerged as important sites for the localization of kDNA replication proteins and minicircle replication intermediates. Previously, we demonstrated that three mitochondrial DNA polymerases (TbPOLIB, TbPOLIC, and TbPOLID) are essential for parasite survival and kDNA replication in both life cycle stages (4)(5)(6)22). While TbPOLIB and TbPOLIC were detected in the KFZ, TbPOLID did not specifically localize near the kDNA disk (22).…”

“…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). However, the initial localization of TbPOLID using a peptide antibody showed that it was distributed throughout the mitochondrial matrix (22), suggesting that POLID would need to redistribute to the kDNA disk to perform its essential role in kDNA replication.…”

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

“…The mitochondrial localization of the Pol ␤ enzymes in T. brucei is in contrast to other eukaryotes, where Pol ␤ enzymes participate in nuclear DNA repair. The three other mitochondrial DNA polymerases of T. brucei (POLIB, POLIC, and POLID) are family A proteins that are most related to prokaryotic DNA polymerase I and appear to function in the earlier stages of kDNA replication, each with a specialized function (4,7,21). 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.…”

“…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.…”