Protein-mediated genome folding allosterically enhances site-specific integration of foreign DNA into CRISPRs

Santiago-Frangos, A.; Henriques, W. S.; Wiegand, T.; Gauvin, C. C.; Buyukyoruk, M.; Graham, A. B.; Wilkinson, R. A.; Triem, L.; Neselu, K.; Eng, E. T.; Lander, G. C.; Wiedenheft, B.

doi:10.1101/2023.05.26.542337

bioRxiv · 10.1101/2023.05.26.542337

Protein-mediated genome folding allosterically enhances site-specific integration of foreign DNA into CRISPRs

Santiago-Frangos, A.·Henriques, W. S.·Wiegand, T.·Gauvin, C. C.·Buyukyoruk, M.·Graham, A. B.·Wilkinson, R. A.·Triem, L.·Neselu, K.·Eng, E. T.·Lander, G. C.·Wiedenheft, B.

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Abstract

Bacteria and archaea acquire resistance to viruses and plasmids by integrating fragments of foreign DNA into the first repeat of a CRISPR array. However, the mechanism of site-specific integration remains poorly understood. Here, we determine a 560 kDa integration complex structure that explains how Cas (Cas1-2/3) and non-Cas proteins (IHF) fold 150 base-pairs of host DNA into a U-shaped bend and a loop that protrude from Cas1-2/3 at right angles. The U-shaped bend traps foreign DNA on one face of the Cas1-2/3 integrase, while the loop places the first CRISPR repeat in the Cas1 active site. Both Cas3s rotate 100-degrees to expose DNA binding sites on either side of the Cas2 homodimer, that each bind an inverted repeat motif in the leader. Leader sequence motifs direct Cas1-2/3-mediated integration to diverse repeat sequences that have a 5-GT.

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Santiago-Frangos, A., Henriques, W. S., Wiegand, T., Gauvin, C. C., Buyukyoruk, M., Graham, A. B., Wilkinson, R. A., Triem, L., Neselu, K., Eng, E. T., Lander, G. C., Wiedenheft, B.. 2023-05-26. Protein-mediated genome folding allosterically enhances site-specific integration of foreign DNA into CRISPRs. https://doi.org/10.1101/2023.05.26.542337

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