TAILIEUCHUNG - Báo cáo khoa học: 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 fromSulfolobus solfataricusstrain IT3 was identified using a structural–functional approach. The N-terminal domain of the pIT3 repli-cation protein encompassing residues 31–245 (. 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. | ỊFEBS Journal Molecular modeling and functional characterization of the monomeric primase-polymerase domain from the Sulfolobus solfataricus plasmid pIT3 Santina Prato1 Rosa Maria Vitale2 Patrizia Contursi1 Georg Lipps3 Michele Saviano4 Mose Rossi1 5 and Simonetta Bartolucci1 1 Dipartimento di Biologia Strutturale e Funzionale Universita degli Studi di Napoli Federico II Naples Italy 2 Istituto di Chimica Biomolecolare CNR Pozzuoli Naples Italy 3 Institute of Biochemistry University of Bayreuth Germany 4 Istituto di Biostrutture e Bioimmagini CNR Naples Italy 5 Istituto di Biochimica delle Proteine CNR Naples Italy Keywords DNA replication pIT3 plasmid primase-polymerase domain Sulfolobus terminal transferase Correspondence S. Bartolucci Dipartimento di Biologia Strutturale e Funzionale University degli Studi di Napoli Federico II Complesso Universitario di Monte S. Angelo Via Cinthia 80126 Naples Italy Fax 39 0816 79053 Tel 39 0816 79052 E-mail bartoluc@ Received 4 April2008 revised 23 June 2008 accepted 4 July 2008 doi 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 . 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. .

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