Flexible tethering of primase and DNA Pol α in the eukaryotic primosome

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Núñez-Ramírez, R 
Klinge, S 
Sauguet, L 
Melero, R 
Recuero-Checa, MA 

The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA-DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA-DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes. The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication.

Amino Acid Sequence, DNA Polymerase I, DNA Primase, Models, Molecular, Molecular Sequence Data, Protein Subunits, RNA, Saccharomyces cerevisiae
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Nucleic Acids Research
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Oxford University Press
Wellcome Trust (084279/Z/07/A)
Wellcome Trust (084279/Z/07/Z)
Spanish Ministry of Science and Innovation (SAF2008-00451 to O.L.); the ‘Red Temática de Investigación Cooperativa en Cáncer (RTICC)’ from the ‘Instituto de Salud Carlos III’ (RD06/0020/1001 to O.L.); the Human Frontiers Science Program (RGP39/2008 to O.L. and E.N.); a Wellcome Trust Senior Fellowship award in Basic Biomedical Sciences (to L.P.); a FPI fellowship from the Spanish Ministry of Science and Innovation to MAR-C; a JAE-DOC contract of the ‘Consejo Superior de Investigaciones Científicas (CSIC)’ and a ‘Juan de la Cierva’ contract from the Spanish Ministry of Science to BGA; EN is a Howard Hughes Medical Institute Investigator. Funding for open access charge: Spanish Ministry of Science and Innovation (SAF2008-00451 to O.L.).