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henriqueswill.bsky.social

@henriqueswill.bsky.social
199 followers 880 following 11 posts

Genetic conflict | Structural biology

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henriqueswill.bsky.social @henriqueswill.bsky.social · 18/06/2025
Full integration requires two sequential transesterification reactions. Focusing on the CRISPR repeat, we learned that the CRISPR repeat is distorted at key conserved purine-pyrimidine steps as it passes from the first transesterification site to the second.
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henriqueswill.bsky.social @henriqueswill.bsky.social · 18/06/2025
We next added short fragments of foreign DNA with and without a protospacer adjacent motif (PAM), to understand why the PAM blocks integration into the CRISPR array. We learned that foreign DNA length alters the complex stability, and that adding 2 bp makes all the difference.
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henriqueswill.bsky.social @henriqueswill.bsky.social · 18/06/2025
We next determined the structure of the complex formed by adding a short foreign DNA fragment to Cas1-2/3. Foreign DNA binding triggers dramatic conformational changes that expose new DNA binding surfaces necessary for homing the integrase to the CRISPR locus.
Cas1-2/3 integrase bound to foreign DNA. The foreign DNA fragment binds to the positively charged channel. The propeller arms formed by the Cas3 lobes rotate out, creating a dogbone shape that exposes three additional DNA-binding channels.
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henriqueswill.bsky.social @henriqueswill.bsky.social · 18/06/2025
Intriguingly, we saw that in this DNA-unbound conformation, a short loop in the Cas3 RecA1 domain covers the nuclease active site, much like a latched gate. Comparing Cas3 in this OFF state to a previously nuclease ON state reveals the gate swings open when the nuclease is active.
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henriqueswill.bsky.social @henriqueswill.bsky.social · 18/06/2025
We wanted to understand how this fantastic genomic knot assembles, so we set out to determine multiple structures of the Cas1-2/3 complex at distinct stages of CRISPR adaptation. In the absence of DNA, Cas1-2/3 forms a prominent, positively charged channel on one face of the complex.
cryoEM structures of the Cas1-2/3 integrase. The heterohexamer assembles into a four-bladed propeller, with one face lined with positively charged residues.
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henriqueswill.bsky.social @henriqueswill.bsky.social · 18/06/2025
New preprint from the Wiedenheft lab! We used #cryoEM to show how a type I-F #CRISPR integrase captures, delivers, and integrates foreign DNA. www.biorxiv.org/content/10.1...
A schematic of a bacterial cell being infected by a phage, with the Cas operon and CRISPR locus detailed. The Cas1-2/3 integrase adopts distinct conformations during the capture, delivery, and integration of a short fragment of the phage DNA.
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