Molecular modeling and functional characterization of the monomeric primase–polymerase domain from the Sulfolobus solfataricus plasmid pIT3

Abstract

In all cell types, chromosomal DNA replication is a complex process entailing three enzymatic activities: helicase activity for double-helix unzipping and primase and DNA polymerase for RNA primer de novo synthesizing and elongation respectively [1,2].Based on the biochemical data accumulated to date, archaeal DNA replication involves a smaller number of polypeptides at each stage of the process and is thus just a simpler form of the much more complex eukaryotic replication machinery [3][4][5][6]. Nonetheless, Archaea are not simply 'mini Eukarya'. A better definition would be 'a mosaic of eukaryal and bacterial systems with specific archaeal features'. Aspects worth mentioning in this respect are the promiscuous nature of the nucleic acid functions performed by archaeal primases and the dual, template-dependent and A tri-functional monomeric primase-polymerase domain encoded by the plasmid pIT3 from Sulfolobus solfataricus strain IT3 was identified using a structural-functional approach. The N-terminal domain of the pIT3 replication protein encompassing residues 31-245 (i.e. Rep245) was modeled onto the crystallographic structure of the bifunctional primase-polymerase domain of the archaeal plasmid pRN1 and refined by molecular dynamics in solution. The Rep245 protein was purified following overexpression in Escherichia coli and its nucleic acid synthesis activity was characterized. The biochemical properties of the polymerase activity such as pH, temperature optima and divalent cation metal dependence were described. Rep245 was capable of utilizing both ribonucleotides and deoxyribonucleotides for de novo primer synthesis and it synthesized DNA products up to several kb in length in a template-dependent manner. Interestingly, the Rep245 primase-polymerase domain harbors also a terminal nucleotidyl transferase activity, being able to elongate the 3¢-end of synthetic oligonucleotides in a non-templated manner. Comparative sequence-structural analysis of the modeled Rep245 domain with other archaeal primase-polymerases revealed some distinctive features that could account for the multifaceted activities exhibited by this domain. To the best of our knowledge, Rep245 typifies the shortest functional domain from a crenarchaeal plasmid endowed with DNA and RNA synthesis and terminal transferase activity.

Dark matter in archaeal genomes: a rich source of novel mobile elements, defense systems and secretory complexes

“…Inspection of the smaller islands revealed yet uncharacterized putative elements that encode a protein (arCOG07809, e.g., Mpal_2033 from Methanosphaerula palustris ) distantly related to the primase-polymerase (prim-pol) domains of replication proteins from plasmids of Sulfolobus (Lipps 2011; Prato et al 2008) and Thermococcus (Gill et al 2014; Krupovic et al 2013) as well as certain bacterial viruses (Halgasova et al 2012). All these proteins are members of the large superfamily of archaeao-eukaryotic primases (AEP) which also includes the catalytic subunit of the archaeal and eukaryotic primases, the enzymes responsible for the initiation of DNA replication (Iyer et al 2005).…”

A highly divergent archaeo-eukaryotic primase from the Thermococcus nautilus plasmid, pTN2

“…Like the heterodimeric DNA polymerase/primase of S. solfataricus (28,29) and the monomeric primase–polymerase domain encoded by the plasmid pIT3 from S. solfataricus strain IT3 (30), PolpTN2 displayed terminal transferase activity. To characterize in more detail this activity, we used two different types of primer-templates, i.e single-stranded DNA and blunt double-stranded DNA, in the presence of Mg 2+ (Figure 6A and B) (see Materials and Methods for details).…”

Metagenomics of microbial and viral life in terrestrial geothermal environments

“…One of the main limitations of this approach is the expression of heterologous genes in E. coli (Gabor et al 2004). Although the commonly used E.coli strains have relaxed requirements for promoter recognition and translation initiation, in this host proteins may not fold correctly and many genes from extremophilic environmental samples are not translated efficiently, especially those belonging to archaea, which might show a codon usage bias (Prato et al 2008). Alternative hosts and broad-host-range vectors may be required to overcome these limitations (Angelov et al 2009;Cheng et al 2014).…”

“…In addition, viral metagenomics and genomics of cultured viruses has also revealed that a large proportion of predicted archaeal viral genes are 'unknown' or 'hypothetical' (Contursi et al 2014a;Prato et al 2008) expanding the content of genetic information referred as biological 'dark matter' (Martinez-Garcia et al 2014). It is expected that the annotation of additional sequenced genomes as well as the exploitation of bioinformatics tools based on structural protein homologies will help to disclose this unexplored repository of viral genes (Fig.…”