Primer synthesis by a eukaryotic-like archaeal primase is independent of its Fe-S cluster.

DNA replication depends on primase, the specialised polymerase responsible for synthesis of the RNA primers that are elongated by the replicative DNA polymerases. In eukaryotic and archaeal replication, primase is a heterodimer of two subunits, PriS and PriL. Recently, a third primase subunit named PriX was identified in the archaeon Sulfolobus solfataricus. PriX is essential for primer synthesis and is structurally related to the Fe-S cluster domain of eukaryotic PriL. Here we show that PriX contains a nucleotide-binding site required for primer synthesis, and demonstrate equivalence of nucleotide-binding residues in PriX with eukaryotic PriL residues that are known to be important for primer synthesis. A primase chimera, where PriX is fused to a truncated version of PriL lacking the Fe-S cluster domain retains wild-type levels of primer synthesis. Our evidence shows that PriX has replaced PriL as the subunit that endows primase with the unique ability to initiate nucleic acid synthesis. Importantly, our findings reveal that the Fe-S cluster is not required for primer synthesis

Structural Basis for Inhibition of Human Primase by Arabinofuranosyl Nucleoside Analogues Fludarabine and Vidarabine.

Nucleoside analogues are widely used in clinical practice as chemotherapy drugs. Arabinose nucleoside derivatives such as fludarabine are effective in the treatment of patients with acute and chronic leukemias and non-Hodgkin's lymphomas. Although nucleoside analogues are generally known to function by inhibiting DNA synthesis in rapidly proliferating cells, the identity of their in vivo targets and mechanism of action are often not known in molecular detail. Here we provide a structural basis for arabinose nucleotide-mediated inhibition of human primase, the DNA-dependent RNA polymerase responsible for initiation of DNA synthesis in DNA replication. Our data suggest ways in which the chemical structure of fludarabine could be modified to improve its specificity and affinity toward primase, possibly leading to less toxic and more effective therapeutic agents.Boehringer-Ingelheim Fonds PhD Fellowship Janggen-Pöhn- Stiftung Award Swiss National Science Foundation Award (all to Sandro Holzer

Threonine 57 is required for the post-translational activation of Escherichia coli aspartate α-decarboxylase.

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formed via the intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation

Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius.

In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.N.P.R. is grateful to the Department of Biochemistry and the Isaac Newton Trust (Trinity College, Cambridge) for supporting this study. The N.P.R. laboratory is funded by the Medical Research Council [Career Development Award G0701443], and also by an Isaac Newton Trust Research Grant. S.M.B. is supported by a BBSRC Doctoral Training Grant [RG53842]. L.P. and M.L.K were funded by a Wellcome Trust Senior Fellowship Award in Basic Biomedical Sciences [grant no. 08279/Z/07/Z]. S.V.A. was supported by intramural funds of the Max Planck Society.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms916

Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with DNA polymerase α-primase.

The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the causative agent of coronavirus disease 2019 (COVID-19)-are under intense current scrutiny to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent proteomic screens of the interactions between viral and host proteins have identified the human proteins targeted by SARS-CoV-2. The DNA polymerase α (Pol α)-primase complex or primosome-responsible for initiating DNA synthesis during genomic duplication-was identified as a target of nonstructural protein 1 (nsp1), a major virulence factor in the SARS-CoV-2 infection. Here, we validate the published reports of the interaction of nsp1 with the primosome by demonstrating direct binding with purified recombinant components and providing a biochemical characterization of their interaction. Furthermore, we provide a structural basis for the interaction by elucidating the cryo-electron microscopy structure of nsp1 bound to the primosome. Our findings provide biochemical evidence for the reported targeting of Pol α by the virulence factor nsp1 and suggest that SARS-CoV-2 interferes with Pol α's putative role in the immune response during the viral infection

Structure of an archaeal PCNA1-PCNA2-FEN1 complex: elucidating PCNA subunit and client enzyme specificity.

The archaeal/eukaryotic proliferating cell nuclear antigen (PCNA) toroidal clamp interacts with a host of DNA modifying enzymes, providing a stable anchorage and enhancing their respective processivities. Given the broad range of enzymes with which PCNA has been shown to interact, relatively little is known about the mode of assembly of functionally meaningful combinations of enzymes on the PCNA clamp. We have determined the X-ray crystal structure of the Sulfolobus solfataricus PCNA1-PCNA2 heterodimer, bound to a single copy of the flap endonuclease FEN1 at 2.9 A resolution. We demonstrate the specificity of interaction of the PCNA subunits to form the PCNA1-PCNA2-PCNA3 heterotrimer, as well as providing a rationale for the specific interaction of the C-terminal PIP-box motif of FEN1 for the PCNA1 subunit. The structure explains the specificity of the individual archaeal PCNA subunits for selected repair enzyme 'clients', and provides insights into the co-ordinated assembly of sequential enzymatic steps in PCNA-scaffolded DNA repair cascades

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

CryoEM insights into RNA primer synthesis by the human primosome.

Publication status: PublishedEukaryotic DNA replication depends on the primosome - a complex of DNA polymerase alpha (Pol α) and primase - to initiate DNA synthesis by polymerisation of an RNA-DNA primer. Primer synthesis requires the tight coordination of primase and polymerase activities. Recent cryo-electron microscopy (cryoEM) analyses have elucidated the extensive conformational transitions required for RNA primer handover between primase and Pol α and primer elongation by Pol α. Because of the intrinsic flexibility of the primosome, however, structural information about the initiation of RNA primer synthesis is still lacking. Here, we capture cryoEM snapshots of the priming reaction to reveal the conformational trajectory of the human primosome that brings DNA primase subunits 1 and 2 (PRIM1 and PRIM2, respectively) together, poised for RNA synthesis. Furthermore, we provide experimental evidence for the continuous association of primase subunit PRIM2 with the RNA primer during primer synthesis, and for how both initiation and termination of RNA primer polymerisation are licenced by specific rearrangements of DNA polymerase alpha catalytic subunit (POLA1), the polymerase subunit of Pol α. Our findings fill a critical gap in our understanding of the conformational changes that underpin the synthesis of the RNA primer by the primosome. Together with existing evidence, they provide a complete description of the structural dynamics of the human primosome during DNA replication initiation

A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome

The DNA polymerase α-primase complex forms an essential part of the eukaryotic replisome. The catalytic subunits of primase and pol α synthesize composite RNA-DNA primers that initiate the leading and lagging DNA strands at replication forks. The physical basis and physiological significance of tethering primase to the eukaryotic replisome via pol α remain poorly characterized. We have identified a short conserved motif at the extreme C terminus of pol α that is critical for interaction of the yeast ortholog pol1 with primase. We show that truncation of the C-terminal residues 1452–1468 of Pol1 abrogates the interaction with the primase, as does mutation to alanine of the invariant amino acid Phe(1463). Conversely, a pol1 peptide spanning the last 16 residues binds primase with high affinity, and the equivalent peptide from human Pol α binds primase in an analogous fashion. These in vitro data are mirrored by experiments in yeast cells, as primase does not interact in cell extracts with pol1 that either terminates at residue 1452 or has the F1463A mutation. The ability to disrupt the association between primase and pol α allowed us to assess the physiological significance of primase being tethered to the eukaryotic replisome in this way. We find that the F1463A mutation in Pol1 renders yeast cells dependent on the S phase checkpoint, whereas truncation of Pol1 at amino acid 1452 blocks yeast cell proliferation. These findings indicate that tethering of primase to the replisome by pol α is critical for the normal action of DNA replication forks in eukaryotic cells