Abstract
We report the characterization of a DNA primase/polymerase protein (PolpTN2) encoded by the pTN2 plasmid from Thermococcus nautilus. Sequence analysis revealed that this protein corresponds to a fusion between an N-terminal domain homologous to the small catalytic subunit PriS of heterodimeric archaeal and eukaryotic primases (AEP) and a C-terminal domain related to their large regulatory subunit PriL. This unique domain configuration is not found in other virus- and plasmid-encoded primases in which PriS-like domains are typically fused to different types of helicases. PolpTN2 exhibited primase, polymerase and nucleotidyl transferase activities and specifically incorporates dNTPs, to the exclusion of rNTPs. PolpTN2 could efficiently prime DNA synthesis by the T. nautilus PolB DNA polymerase, suggesting that it is used in vivo as a primase for pTN2 plasmid replication. The N-terminal PriS-like domain of PolpTN2 exhibited all activities of the full-length enzyme but was much less efficient in priming cellular DNA polymerases. Surprisingly, the N-terminal domain possesses reverse transcriptase activity. We speculate that this activity could reflect an ancestral function of AEP proteins in the transition from the RNA to the DNA world.
Highlights
Genes encoding DNA-dependent DNA polymerases are present in all cellular genomes and in genomes of many double-stranded DNA viruses
The RNA primers are synthesized by specific DNA-dependent RNA polymerases called DNA primases, which are ubiquitous in the living world
The results suggested that PolpTN2 consists of two domains corresponding to the PriS and PriL domains, respectively, of heterodimeric archaeo-eukaryotic primase (AEP) primases (Figure 1A)
Summary
Genes encoding DNA-dependent DNA polymerases (hereafter referred to as DNA polymerases) are present in all cellular genomes (with often several functional analogues and/or paralogues) and in genomes of many double-stranded DNA viruses. Most of these DNA polymerases require a free 3’OH borne by DNA strands or RNA primers to initiate DNA synthesis. In 2003, Georg Lipps identified a DNA polymerase/ primase-encoding gene in a small cryptic plasmid, pRN1, from the thermophilic archaeon, Sulfolobus islandicus [2] This DNA polymerase/primase can prime DNA synthesis on a single-stranded template without addition of a 3’OH-containing oligonucleotide as primer, indicating that it can synthesize a ‘DNA primer’. Further sequence analyses have shown that these proteins belong to a wide superfamily of proteins, which was termed the archaeo-eukaryotic primase (AEP) superfamily [4]
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