Prespacers formed during primed adaptation associate with the Cas1–Cas2 adaptation complex and the Cas3 interference nuclease–helicase
| Autor: | Ekaterina Savitskaya, Anna Shiriaeva, Olga Musharova, Evgeny Klimuk, Noemí M. Guzmán, Ekaterina Semenova, Sofia Medvedeva, Victor G. Zgoda, Konstantin Severinov, Daria Titova |
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| Rok vydání: | 2021 |
| Předmět: |
CRISPR interference
Multidisciplinary biology Effector Chemistry Escherichia coli Proteins DNA Helicases RNA Priming (immunology) Helicase Biological Sciences Endonucleases Microbiology CRISPR adaptation Integrase Cell biology Escherichia coli biology.protein CRISPR CRISPR-Cas Systems CRISPR-Cas Chromatin immunoprecipitation |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | Significance Primed adaptation allows rapid acquisition of protective spacers derived from foreign mobile genetic elements into CRISPR arrays of the host. Primed adaptation requires ongoing CRISPR interference that destroys foreign genetic elements, but the nature of this requirement is unknown. Using the Escherichia coli I-E CRISPR-Cas as a model, we show that prespacers, short fragments of foreign DNA on their way to become incorporated into CRISPR arrays as spacers, are associated with both the adaptation integrase Cas1 and the interference nuclease Cas3, implying physical association of the interference and adaptation machineries during priming. For Type I CRISPR-Cas systems, a mode of CRISPR adaptation named priming has been described. Priming allows specific and highly efficient acquisition of new spacers from DNA recognized (primed) by the Cascade-crRNA (CRISPR RNA) effector complex. Recognition of the priming protospacer by Cascade-crRNA serves as a signal for engaging the Cas3 nuclease–helicase required for both interference and primed adaptation, suggesting the existence of a primed adaptation complex (PAC) containing the Cas1–Cas2 adaptation integrase and Cas3. To detect this complex in vivo, we here performed chromatin immunoprecipitation with Cas3-specific and Cas1-specific antibodies using cells undergoing primed adaptation. We found that prespacers are bound by both Cas1 (presumably, as part of the Cas1–Cas2 integrase) and Cas3, implying direct physical association of the interference and adaptation machineries as part of PAC. |
| Databáze: | OpenAIRE |
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