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 β during replication

Abstract

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 f lagellum. 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 ␤ at antipodal sites f lanking 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 The trypanosomatid Crithidia fasciculata has an unusual mitochondrial DNA structure called the kinetoplast DNA, which is composed of 5,000 minicircles of 2.5 kilobases (kb) and 25 maxicircles of 37 kb catenated into a single network. The maxicircles encode conventional mitochondrial proteins and ribosomal RNAs. Unlike most mitochondrial DNA, kinetoplast DNA replicates only once in the cell cycle in approximate synchrony with nuclear DNA replication (1, 2). Minicircles replicate free of the network as intermediates (3). Lightstrand synthesis is RNA primed at each of the two universal minicircle sequences and occurs continuously (4-6). Heavystrand synthesis occurs discontinuously via Okazaki-like fragments that are also likely to be RNA primed (5, 7). Nicks and gaps remaining in newly replicated minicircles are partially repaired before reattachment to the network but are not completely repaired until just before network division into two daughter networks. (4,5,8,9).Several enzymes involved in kinetoplast DNA replication have been purified and localized within the kinetoplast. A primase has been localized to a region adjacent to the two faces of the kinetoplast disk, suggesting that RNA priming and the early stages of minicircle replication may occur in this region (10). Additionally, a topoisomerase II and a DNA polymerase ␤ have been localized to two antipodal sites flanking the kinetoplast disc where the minicircles are reattached to the network (11-14). Minicircle replication intermediates have also been detected at these sites (11). The low processivity of polymerase ␤ suggests that it may be involved in the repair of gaps remaining in newly replicated minicircles. The topoisomerase II is likely to mediate minicircle reattachment after partial gap repair.To further define the components of the replication machinery and their localization relative to the kinetoplast disc, we have sought to identify and purify additional kinetoplast replication proteins. The purification and characterization of a structure-specific endonuclease (SSE1) enriched in the kinetoplast fraction of C. fasciculata ...

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

“…Redistribution was proposed as a mechanism for the localization to the antipodal sites. However, these proteins were undetectable by IF when not present at the antipodal sites, even though protein levels remained constant (12,20). Here, we provide strong evidence that TbPOLID antipodal localization is due to dynamic redistribution from the mitochondrial matrix in a time-dependent manner during kDNA synthesis.…”

“…Interestingly, several antipodal site proteins appear to transiently associate with this region, demonstrating that the protein composition is dynamic (20,43,46). Studies from the related trypanosome Crithidia fasiculata established that the antipodal localization of Pol ␤ and SSE1 correlated with kDNA synthesis and not other cell cycle stages (12,20). Redistribution was proposed as a mechanism for the localization to the antipodal sites.…”

“…Multiple lines of evidence support this view. First, Okazaki fragment-processing enzymes Pol ␤ and SSE1 appear to colocalize (12), while TopoII mt and Ligk ␤ do not precisely colocalize (9). Second, ethanolic-phosphotungstic acid staining showed regions that differ in staining intensity, indicating the variability of protein concentration in different regions of the antipodal sites (15).…”

“…Originally, the Okazaki fragment-processing proteins, Pol ␤ and SSE1, as well as TopoII mt , were localized to the antipodal sites (12,13,32). A great majority of newly identified essential kDNA replication proteins with roles in origin binding, priming, and processive replication also localize to the antipodal sites (p38, p93, PRI1, PRI2, PIF1, and PIF5), suggesting that the role of the antipodal sites is not limited to Okazaki fragment processing.…”

“…Minicircle progeny (still containing at least one gap) are subsequently reattached at two electron-dense areas positioned 180°apart at the network periphery called the antipodal sites. Several proteins associated with Okazaki fragment processing and reattachment to the network localize to the antipodal sites, including SSE1, DNA Pol ␤, DNA ligase k␤ (Ligk ␤), and topoisomerase II (Topo II mt ) (9,12,20,43,50). As a result, there is spatial and temporal separation of replication events with early initiation occurring in the KFZ, followed by Okazaki fragment processing and reattachment at the antipodal sites.…”