Structure reveals why genome folding is necessary for site-specific integration of foreign DNA into CRISPR arrays
- Santiago-Frangos, Andrew [ Montana State University: Microbiology & Cell Biology ]
- Henriques, William S. [ Montana State University: Microbiology & Cell Biology ]
- Wiegand, Tanner [ Montana State University: Microbiology & Cell Biology ]
- Gauvin, Colin C. [ Montana State University: Chemistry & Biochemistry ]
- Buyukyoruk, Murat [ Montana State University: Microbiology & Cell Biology ]
- Graham, Ava B. [ Montana State University: Microbiology & Cell Biology ]
- Wilkinson, Royce A. [ Montana State University: Microbiology & Cell Biology ]
- Triem, Lenny [ Montana State University: Microbiology & Cell Biology ]
- Neselu, Kasahun
- Eng, Edward T.
- Lander, Gabriel C.
- Wiedenheft, Blake [ Montana State University: Microbiology & Cell Biology ]
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 Pseudomonas aeruginosa Cas (Cas1–Cas2/3) and non-Cas proteins (for example, integration host factor) 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 Cas3 proteins rotate 100 degrees to expose DNA-binding sites on either side of the Cas2 homodimer, which 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. Collectively, this work reveals new DNA-binding surfaces on Cas2 that are critical for DNA folding and site-specific delivery of foreign DNA.