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International audience; Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323–785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements.

13 páginas, 7 figuras -- PAGS nros. 8187-8199, Núñez-Ramírez, Rafael et al., 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, 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.)

International audience; Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication.

Background DNA synthesis during replication relies on RNA primers synthesised by the primase, a specialised DNA-dependent RNA polymerase that can initiate nucleic acid synthesis de novo. In archaeal and eukaryotic organisms, the primase is a heterodimeric enzyme resulting from the constitutive association of a small (PriS) and large (PriL) subunit. The ability of the primase to initiate synthesis of an RNA primer depends on a conserved Fe-S domain at the C-terminus of PriL (PriL-CTD). However, the critical role of the PriL-CTD in the catalytic mechanism of initiation is not understood. Methodology/Principal Findings Here we report the crystal structure of the yeast PriL-CTD at 1.55 A resolution. The structure reveals that the PriL-CTD folds in two largely independent alpha-helical domains joined at their interface by a [4Fe-4S] cluster. The larger N-terminal domain represents the most conserved portion of the PriL-CTD, whereas the smaller C-terminal domain is largely absent in archaeal PriL. Unexpectedly, the N-terminal domain reveals a striking structural similarity with the active site region of the DNA photolyase/cryptochrome family of flavoproteins. The region of similarity includes PriL-CTD residues that are known to be essential for initiation of RNA primer synthesis by the primase. Conclusion/Significance Our study reports the first crystallographic model of the conserved Fe-S domain of the archaeal/eukaryotic primase. The structural comparison with a cryptochrome protein bound to flavin adenine dinucleotide and single-stranded DNA provides important insight into the mechanism of RNA primer synthesis by the primase.

International audience; The immunopurified yeast DNA polymerase--DNA primase complex is constituted by DNA polymerase I polypeptides and by three other protein species, called p74, p58 and p48, which we show to be immunologically unrelated. The gene encoding the p48 polypeptide has been identified by immunological screening of a lambda gt11 yeast genomic DNA library. Antiserum specific for p48 inhibits DNA primase, and immunoreactive, inhibitory antibodies are affinity-purified by the clone-encoded protein, thus relating the p48 polypeptide to DNA primase activity. The entire gene has been cloned, and the 1.45-kb p48 mRNA is overproduced in cells containing the gene in high copy number. Gene disruption and Southern hybridization experiments demonstrate that the p48 protein is encoded by a single gene and it performs an essential function.