Properties of an unusual DNA primase from an archaeal plasmid

Abstract

Primases are specialized DNA-dependent RNA polymerases that synthesize a short oligoribonucleotide complementary to single-stranded template DNA. In the context of cellular DNA replication, primases are indispensable since DNA polymerases are not able to start DNA polymerization de novo.The primase activity of the replication protein from the archaeal plasmid pRN1 synthesizes a rather unusual mixed primer consisting of a single ribonucleotide at the 5′ end followed by seven deoxynucleotides. Ribonucleotides and deoxynucleotides are strictly required at the respective positions within the primer. Furthermore, in contrast to other archaeo-eukaryotic primases, the primase activity is highly sequence-specific and requires the trinucleotide motif GTG in the template. Primer synthesis starts outside of the recognition motif, immediately 5′ to the recognition motif. The fidelity of the primase synthesis is high, as non-complementary bases are not incorporated into the primer.

Identification of the minimal replicon and the origin of replication of the crenarchaeal plasmid pRN1

“…This operon consists of the two co-transcribed genes -orf56 and orf904. Orf904 codes for the replication protein of pRN1 (Lipps et al 2003;Beck and Lipps 2007), whereas ORF56 is involved in plasmid copy number control . It has previously been shown that an interruption of the replication operon abrogates the replication of pRN1 ).…”

Characterization of a Functional DnaG-Type Primase in Archaea: Implications for a Dual-Primase System

“…PriSL has also been shown to possess a terminal transferase activity to extend ssDNA substrates. 4,5,47 SsoDnaG is a more robust primase, with the k cat for RNA primer formation being roughly fourfold higher than that for SsoPriSL. Interestingly, the enzymatic performance constants (k cat /K m ) are roughly equal (Table 1).…”

Archaeal Extrachromosomal Genetic Elements

“…Georg Lipps and colleagues characterized all three conserved replication proteins encoded in pRN1, including the copy number regulation protein CopG (encoded by ORF56) (219), a DNA-binding protein implicated in plasmid maintenance (PlrA) (encoded by ORF80) (154), and a multifunctional replication protein (RepA) (159,(220)(221)(222). They were the first to reveal that both primase and DNA polymerase (Prim/Pol) activities resided in the pRN1 RepA protein and localized the Prim/Pol activities to the N-terminal part of RepA (220,221). They also found that the C-terminal helicase domain is structurally similar to a superfamily 3 helicase, displaying weak helicase activity in vitro (159).…”