Primases synthesise the RNA primers that are necessary for replication of the parental DNA strands. Here we report that the heterodimeric archaeal/eukaryotic primase is an iron-sulfur (Fe-S) protein. Binding of the Fe-S cluster is mediated by an evolutionarily conserved domain at the C terminus of the large subunit. We further show that the Fe-S domain is essential to the unique ability of the eukaryotic primase to start DNA replication.De novo synthesis of RNA primers by primases is essential for cellular and viral DNA replication1,2. Archaeal and eukaryotic primases are heterodimeric enzymes with a small (PriS) and a large (PriL) subunit2. Although the catalytic activity resides within PriS, the PriL subunit is necessary to primase function as disruption of the PriL gene in yeast is lethal3. Reported roles for PriL include stabilisation of PriS, participation in initiation of RNA primer synthesis, determination of product size and transfer of the primer to DNA polymerase α4-11. A recent crystallographic model of the heterodimeric primase from the archaeon Sulfolobus solfataricus provided the first description of the large subunit but did not include its C-terminal domain (PriL-CTD)12. The presence of four conserved cysteines in archaeal and eukaryotic PriL-CTD sequences suggests that the PriL-CTD might be a metal-binding domain (Supplementary Figure 1).We set out to characterise the biochemical and biophysical properties of the PriL-CTD. Freshly purified samples of S. solfataricus PriL-CTD expressed in bacteria as glutathione Stransferase (GST) fusion protein consistently displayed a yellow-brown colour, which turned darker upon concentration of the sample (Supplementary Figure 2). The absorption spectrum of the S. solfataricus GST-PriLCTD showed a broad shoulder around 400 nanometers (nm), next to the expected protein peak at 280 nm (Figure 1a). Fading of the colour under aerobic conditions and a decrease in absorption at 400 nm over time indicated the presence of a chromophore in the PriL-CTD, which is sensitive to air oxidation. Purified Saccharomyces cerevisiae PriL-CTD fused to a maltose-binding protein (MBP) displayed a similar colour and increased absorption at 400 nm as the S. solfataricus GST-PriLCTD (Supplementary * Correspondence (luca@cryst.bioc.cam.ac.uk). Author Contributions SK and LP conceived the project and designed the experiments; SK prepared the recombinant proteins and performed the biochemical experiments; JH performed the EPR analysis; JDM first suggested that the chromophore in the PriL-CTD might be a Fe-S cluster and performed the CD analysis; TK assisted with the primase assays; SK and LP interpreted the data and wrote the paper.

The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment.DOI: http://dx.doi.org/10.7554/eLife.00482.001

Helicase–nuclease systems dedicated to DNA end resection in preparation for homologous recombination (HR) are present in all kingdoms of life. In thermophilic archaea, the HerA helicase and NurA nuclease cooperate with the highly conserved Mre11 and Rad50 proteins during HR-dependent DNA repair. Here we show that HerA and NurA must interact in a complex with specific subunit stoichiometry to process DNA ends efficiently. We determine crystallographically that NurA folds in a toroidal dimer of intertwined RNaseH-like domains. The central channel of the NurA dimer is too narrow for double-stranded DNA but appears well suited to accommodate one or two strands of an unwound duplex. We map a critical interface of the complex to an exposed hydrophobic epitope of NurA abutting the active site. Based upon the presented evidence, we propose alternative mechanisms of DNA end processing by the HerA-NurA complex.

The successful completion of meiosis is essential for all sexually reproducing organisms. The synaptonemal complex (SC) is a large proteinaceous structure that holds together homologous chromosomes during meiosis, providing the structural framework for meiotic recombination and crossover formation. Errors in SC formation are associated with infertility, recurrent miscarriage and aneuploidy. The current lack of molecular information about the dynamic process of SC assembly severely restricts our understanding of its function in meiosis. Here, we provide the first biochemical and structural analysis of an SC protein component and propose a structural basis for its function in SC assembly. We show that human SC proteins SYCE2 and TEX12 form a highly stable, constitutive complex, and define the regions responsible for their homotypic and heterotypic interactions. Biophysical analysis reveals that the SYCE2–TEX12 complex is an equimolar hetero-octamer, formed from the association of an SYCE2 tetramer and two TEX12 dimers. Electron microscopy shows that biochemically reconstituted SYCE2–TEX12 complexes assemble spontaneously into filamentous structures that resemble the known physical features of the SC central element (CE). Our findings can be combined with existing biological data in a model of chromosome synapsis driven by growth of SYCE2–TEX12 higher-order structures within the CE of the SC.

BackgroundDNA 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 FindingsHere we report the crystal structure of the yeast PriL-CTD at 1.55 Å 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/SignificanceOur 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.