tailieunhanh - Báo cáo khoa học: Molecular basis for the subunit assembly of the primase from an archaeon Pyrococcus horikoshii

Archaeal⁄eukaryotic primases form a heterodimer consisting of a small cat-alytic subunit (PriS) and a large subunit (PriL). The heterodimer complex synthesizes primer oligoribonucleotides that are required for chromosomal replication. Here, we describe crystallographic and biochemical studies of the N-terminal domain (NTD) of PriL (PriL NTD ; residues 1–222) that bind to PriS from a hyperthermophilic archaeon, Pyrococcus horikoshii, at A ˚ resolution. | ễFEBS Journal Molecular basis for the subunit assembly of the primase from an archaeon Pyrococcus horikoshii Nobutoshi Ito1 2 Ikuo Matsui3 and Eriko Matsui3 1 Cellular Physiology Laboratory Discovery Research Institute RIKEN Wako Saitama Japan 2 Division of BiofunctionalScience Tohoku University BiomedicalEngineering Research Organization Sendai Japan 3 BiologicalInformation Research Center NationalInstitute of Advanced IndustrialScience and Technology Ibaraki Japan Keywords Archaea primase Pyrococcus horikoshii replication X-ray crystallography Correspondence I. Matsui Biological Information Research Center NationalInstitute of Advanced IndustrialScience and Technology 1-1 Higashi Tsukuba Ibaraki 305-8566 Japan Fax 81 298 616151 Tel 81 298 616142 E-mail ik-matsui@ Database The atomic coordinates and structure factors have been deposited in the Protein Data Bank accession code 2DLA Research Collaboratory for Structural Bioinformatics Rutgers the State University of New Jersey New Brunswick NJ http . org . Archaeal eukaryotic primases form a heterodimer consisting of a small catalytic subunit PriS and a large subunit PriL . The heterodimer complex synthesizes primer oligoribonucleotides that are required for chromosomal replication. Here we describe crystallographic and biochemical studies of the N-terminal domain NTD of PriL PriLNTD residues 1-222 that bind to PriS from a hyperthermophilic archaeon Pyrococcus horikoshii at A resolution. The PriLNTD structure consists of two subdomains the helixbundle and twisted-strand domains. The latter is structurally flexible and is expected to contain a PriS interaction site. Pull-down and surface plasmon resonance analyses of structure-based deletion and alanine scanning mutants showed that the conserved hydrophobic Tyr155-Tyr156-Ile157 region near the flexible region is the PriS-binding site as the Y155A Y156A I157A mutation markedly reduces PriS binding by 1000-fold. These findings and a structural .