Graphical Abstract Highlights d Liquid-and solid-state NMR structure of a primase HBD bound to DNA and two ATPs d ATP binding triggers the sequence-specific DNA recognition of the primase HBD d In binding nucleotides and template, the HBD prepares dinucleotide synthesis d This preparatory role of the HBD is conserved in archaeoeukaryotic primases
DNA replication is a crucial stage in the transfer of genetic information from parent to daughter cells. This mechanism involves multiple proteins with one key player being the primase. Primases are single-stranded DNA dependent RNA polymerases. On the leading strand, they synthesize the primer once allowing DNA elongation while on the lagging strand primers are generated repeatedly (Okazaki fragments). Primases have the unique ability to create the first phosphodiester bond yielding a dinucleotide which is initially elongated by primases and then by DNA polymerases.Primase activity has been studied in the last decades but the detailed molecular steps explaining some unique features remain unclear. High-resolution structures of free and bound primases domains have brought significant insights in the understanding of the primase reaction cycle. Here, we give a short review of the structural work conducted in the field of archaeo-eukaryotic primases and we underline the missing “pictures” of the active forms of the enzyme which are of major interest. We organized our analysis with respect to the progression through the catalytic pathway.
Priming of single stranded templates is essential for DNA replication. In recent years, significant progress was made in understanding how DNA primase fulfils this fundamental function, particularly with regard to the initiation. Equally intriguing is the unique property of archeao-eukaryotic primases to terminate primer formation at a well-defined unit length. The apparent ability to “count” the number of bases incorporated prior to primer release is not well understood, different mechanisms having been proposed for different species. We report a mechanistic investigation of primer termination by the pRN1 primase from Sulfolobus islandicus. Using an HPLC-based assay we determined structural features of the primer 5′-end that are required for consistent termination. Mutations within the unstructured linker connecting the catalytic domain to the template binding domain allowed us to assess the effect of altered linker length and flexibility on primer termination.
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