Molecular modeling and functional characterization of the monomeric primase-polymerase domain from the Sulfolobus solfataricus plasmid pIT3.
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.
Prato
Santina
S
Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi di Napoli Federico II, Naples, Italy.
Vitale
Rosa Maria
RM
Contursi
Patrizia
P
Lipps
Georg
G
Saviano
Michele
M
Rossi
Mosé
M
Bartolucci
Simonetta
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D019915:DNA Primase / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids; D017386:Sequence Homology, Amino Acid; D048229:Sulfolobus solfataricus / Q000235:genetics*
D017931:DNA Primers; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase