Purification and characterization of organellar DNA polymerases in the red alga Cyanidioschyzon merolae

Abstract

DNA polymerase γ, a mitochondrial replication enzyme of yeasts and animals, is not present in photosynthetic eukaryotes. Recently, DNA polymerases with distant homology to bacterial DNA polymerase I were reported in rice, Arabidopsis, and tobacco, and they were localized to both plastids and mitochondria. We call them plant organellar DNA polymerases (POPs). However, POPs have never been purified in the native form from plant tissues. The unicellular thermotrophic red alga Cyanidioschyzon merolae contains two genes encoding proteins related to Escherichia coli DNA polymerase I (PolA and PolB). Phylogenetic analysis revealed that PolB is an ortholog of POPs. Nonphotosynthetic eukaryotes also have POPs, which suggested that POPs have an ancient origin before eukaryotic photosynthesis. PolA is a homolog of bacterial DNA polymerase I and is distinct from POPs. PolB was purified from the C. merolae cells by a series of column chromatography steps. Recombinant protein of PolA was also purified. Sensitivity to inhibitors of DNA synthesis was different in PolA, PolB, and E. coli DNA polymerase I. Immunoblot analysis and targeting studies with green fluorescent protein fusion proteins demonstrated that PolA was localized in the plastids, whereas PolB was present in both plastids and mitochondria. The expression of PolB was regulated by the cell cycle. The available results suggest that PolB is involved in the replication of plastids and mitochondria.

Characterization of cell-cycle-driven and light-driven gene expression in a synchronous culture system in the unicellular rhodophyte Cyanidioschyzon merolae

“…These characteristics, as well as phylogenetic analysis (Nozaki et al, 2007), suggested that C. merolae is one of the most primitive red algae, probably diverged from a point near the root of the eukaryotes. We also reported a unique pathway of lipid biosynthesis in this alga based on genomic and experimental analyses (Sato & Moriyama, 2007) and the DNA polymerase localized to both plastid and mitochondrion (Moriyama et al, 2008). We proposed to name this new type of organellar DNA polymerase POP (plant organellar DNA polymerase).…”

The HU Protein Is Important for Apicoplast Genome Maintenance and Inheritance in Toxoplasma gondii

“…Both apicomplexan orthologs have been shown to possess polymerase activity and have been suggested to be the replicative enzyme of the apicoplast genome, but whether it is essential for the parasite growth has not yet been demonstrated. In T. gondii there is currently no other good candidate for the replicative complex of the apicoplast DNA, but in Plasmodium there appears to be another homolog of DNA Pol I which may have an organellar localization (32). A homolog of SSB (single-stranded DNA-binding protein) which localizes to the apicoplast and binds single-stranded DNA has also been identified in Apicomplexa (36).…”

Analysis of triacylglycerol accumulation under nitrogen deprivation in the red alga Cyanidioschyzon merolae

“…The unicellular rhodophyte Cyanidioschyzon merolae, originally isolated from an Italian acidic hot spring (De Luca et al, 1978), can be cultivated in the places where agricultural use is difficult, because it grows vigorously at high temperatures (up to 50 uC) (Moriyama et al, 2008) and at acidic pH from 1.5 to 2.5. These growth conditions do not allow the growth of most ordinary contaminants.…”