- MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell genetics MeSH
- DNA-Directed DNA Polymerase genetics metabolism MeSH
- DNA Primase genetics metabolism MeSH
- Extracellular Matrix metabolism pathology MeSH
- Genes, p53 genetics MeSH
- Glioblastoma metabolism pathology MeSH
- Pericytes metabolism pathology MeSH
- BRCA1 Protein genetics metabolism MeSH
- Publication type
- Overall MeSH
During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity1-3. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.
CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.
- MeSH
- Bacteria enzymology genetics MeSH
- Bacteroidetes enzymology genetics MeSH
- Bacterial Proteins genetics metabolism MeSH
- CRISPR-Associated Proteins metabolism MeSH
- CRISPR-Cas Systems * MeSH
- Dimerization MeSH
- DNA Primers biosynthesis MeSH
- DNA-Directed DNA Polymerase genetics metabolism MeSH
- DNA Primase genetics metabolism MeSH
- Escherichia coli metabolism MeSH
- Gene Expression MeSH
- Phylogeny MeSH
- Mutation MeSH
- Prokaryotic Cells metabolism MeSH
- Recombinant Proteins MeSH
- Ribonucleotides metabolism MeSH
- Computational Biology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
We report the crystal structure of the SARS-CoV-2 putative primase composed of the nsp7 and nsp8 proteins. We observed a dimer of dimers (2:2 nsp7-nsp8) in the crystallographic asymmetric unit. The structure revealed a fold with a helical core of the heterotetramer formed by both nsp7 and nsp8 that is flanked with two symmetry-related nsp8 β-sheet subdomains. It was also revealed that two hydrophobic interfaces one of approx. 1340 Å2 connects the nsp7 to nsp8 and a second one of approx. 950 Å2 connects the dimers and form the observed heterotetramer. Interestingly, analysis of the surface electrostatic potential revealed a putative RNA binding site that is formed only within the heterotetramer.
- MeSH
- Betacoronavirus chemistry MeSH
- DNA Primase chemistry metabolism MeSH
- Protein Conformation MeSH
- Crystallography, X-Ray MeSH
- Models, Molecular MeSH
- Protein Multimerization MeSH
- Multiprotein Complexes MeSH
- RNA metabolism MeSH
- Binding Sites MeSH
- Viral Nonstructural Proteins chemistry metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
