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TitleStructure reveals why genome folding is necessary for site-specific integration of foreign DNA into CRISPR arrays.
Journal, issue, pagesNat Struct Mol Biol, Vol. 30, Issue 11, Page 1675-1685, Year 2023
Publish dateSep 14, 2023
AuthorsAndrew Santiago-Frangos / William S Henriques / Tanner Wiegand / Colin C Gauvin / Murat Buyukyoruk / Ava B Graham / Royce A Wilkinson / Lenny Triem / Kasahun Neselu / Edward T Eng / Gabriel C Lander / Blake Wiedenheft /
PubMed AbstractBacteria 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 ...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.
External linksNat Struct Mol Biol / PubMed:37710013 / PubMed Central
MethodsEM (single particle)
Resolution3.48 Å
Structure data

29280

8flj

EMDB-29280, PDB-8flj:
Cas1-Cas2/3 integrase and IHF bound to CRISPR leader, repeat and foreign DNA
Method: EM (single particle) / Resolution: 3.48 Å

Source
  • pseudomonas aeruginosa pa14 (bacteria)
  • synthetic construct (others)
KeywordsRECOMBINATION / DNA BINDING PROTEIN / HYDROLASE/DNA / DNA integration / maintenance of CRISPR repeat elements / IHF-DNA complex / Enzymes altering nucleic acid conformation / Site specific endodeoxyribonucleases: cleavage is not sequence specific / Nucleotidyltransferases / GO:0008301 / HYDROLASE-DNA complex

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