Abstract
Cellular organisms in different domains of life employ structurally unrelated, non-homologous DNA primases for synthesis of a primer for DNA replication. Archaea and eukaryotes encode enzymes of the archaeo-eukaryotic primase (AEP) superfamily, whereas bacteria uniformly use primases of the DnaG family. However, AEP genes are widespread in bacterial genomes raising questions regarding their provenance and function. Here, using an archaeal primase–polymerase PolpTN2 encoded by pTN2 plasmid as a seed for sequence similarity searches, we recovered over 800 AEP homologs from bacteria belonging to 12 highly diverse phyla. These sequences formed a supergroup, PrimPol-PV1, and could be classified into five novel AEP families which are characterized by a conserved motif containing an arginine residue likely to be involved in nucleotide binding. Functional assays confirm the essentiality of this motif for catalytic activity of the PolpTN2 primase–polymerase. Further analyses showed that bacterial AEPs display a range of domain organizations and uncovered several candidates for novel families of helicases. Furthermore, sequence and structure comparisons suggest that PriCT-1 and PriCT-2 domains frequently fused to the AEP domains are related to each other as well as to the non-catalytic, large subunit of archaeal and eukaryotic primases, and to the recently discovered PriX subunit of archaeal primases. Finally, genomic neighborhood analysis indicates that the identified AEPs encoded in bacterial genomes are nearly exclusively associated with highly diverse integrated mobile genetic elements, including integrative conjugative plasmids and prophages.
Highlights
In all cellular organisms, replicative DNA polymerases cannot initiate DNA synthesis de novo
The diversity and abundance of archaeo-eukaryotic primases (AEPs) homologs in bacteria is rather astounding, especially considering that AEPs belong to the core DNA replication machinery of archaea and eukaryotes, but not bacteria, which instead use DnaG family primases
In this study, using an archaeal primase–polymerase PolpTN2 as a seed for sequence similarity searches, we recovered over 800 AEP homologs in bacteria from 12 highly diverse phyla, including those overrepresented in genomic databases, such as Proteobacteria, Firmicutes, and Actinobacteria, and those from less sampled phyla, such as Deinococcus– Thermus, Fusobacteria, and Verrucomicrobia
Summary
Replicative DNA polymerases cannot initiate DNA synthesis de novo. Archaea and eukaryotes encode homologous heterodimeric primases evolutionarily unrelated to DnaG and consisting of the small catalytic (PriS) and the large regulatory (PriL) subunits. The former contains a highly derived version of the RNA recognition motif fold, which is present in viral RNA-dependent RNA polymerases, reverse transcriptases, cyclases, and DNA polymerases of the A/B/Y families [2,3], whereas the PriL consists of two largely α-helical domains, where the C-terminal domain contains a 4Fe–4S cluster [4,5,6]. Archaeo-eukaryotic primases (AEPs) are not directly involved in bacterial DNA replication, many bacteria encode AEP homologs, some of which (http://creativecommons.org/licenses/by/4.0/)
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