Primase
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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 enzymologie genetika MeSH
- Bacteroidetes enzymologie genetika MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Cas proteiny metabolismus MeSH
- CRISPR-Cas systémy * MeSH
- dimerizace MeSH
- DNA primery biosyntéza MeSH
- DNA-dependentní DNA-polymerasy genetika metabolismus MeSH
- DNA-primasa genetika metabolismus MeSH
- Escherichia coli metabolismus MeSH
- exprese genu MeSH
- fylogeneze MeSH
- mutace MeSH
- prokaryotické buňky metabolismus MeSH
- rekombinantní proteiny MeSH
- ribonukleotidy metabolismus MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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 chemie MeSH
- DNA-primasa chemie metabolismus MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- multiproteinové komplexy MeSH
- RNA metabolismus MeSH
- vazebná místa MeSH
- virové nestrukturální proteiny chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
... recommendations for TP53 mutation analysis in chronic lymphocytic leukemia – 2024 update -- DNA polymerase α-primase ...
- MeSH
- chronická lymfatická leukemie genetika MeSH
- DNA-dependentní DNA-polymerasy genetika metabolismus MeSH
- DNA-primasa genetika metabolismus MeSH
- extracelulární matrix metabolismus patologie MeSH
- geny p53 genetika MeSH
- glioblastom metabolismus patologie MeSH
- pericyty metabolismus patologie MeSH
- protein BRCA1 genetika metabolismus MeSH
- Publikační typ
- souhrny MeSH
Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome, caused by genetic mutations that principally affect telomere biology. Approximately 35% of cases remain uncharacterised at the genetic level. To explore the genetic landscape, we conducted genetic studies on a large collection of clinically diagnosed cases of DC as well as cases exhibiting features resembling DC, referred to as 'DC-like' (DCL). This led us to identify several novel pathogenic variants within known genetic loci and in the novel X-linked gene, POLA1. In addition, we have also identified several novel variants in POT1 and ZCCHC8 in multiple cases from different families expanding the allelic series of DC and DCL phenotypes. Functional characterisation of novel POLA1 and POT1 variants, revealed pathogenic effects on protein-protein interactions with primase, CTC1-STN1-TEN1 (CST) and shelterin subunit complexes, that are critical for telomere maintenance. ZCCHC8 variants demonstrated ZCCHC8 deficiency and signs of pervasive transcription, triggering inflammation in patients' blood. In conclusion, our studies expand the current genetic architecture and broaden our understanding of disease mechanisms underlying DC and DCL disorders.
- MeSH
- dítě MeSH
- dyskeratosis congenita * genetika MeSH
- lidé MeSH
- mutace MeSH
- proteiny vázající telomery * genetika metabolismus MeSH
- shelterinový komplex MeSH
- telomery * genetika metabolismus MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
... Huml) -- Staroměstští primátoři (primasové) vletech 1537-1783 (F. ...
418 s. : il.
Encyklopedická příručka podává chronologický přehled jednotlivých dat důležitých pro hospodářský, sociální, politický a kulturní vývoj města do konce roku 1987.
- Klíčová slova
- dějiny, Praha v datech,
- NLK Obory
- humanitní vědy a umění
