5xvo | pdb_00005xvo
E. fae Cas1-Cas2/prespacer/target ternary complex revealing DNA sampling and half-integration states
Structural highlights
Function[E6GPD7_ENTFL] CRISPR (clustered regularly interspaced short palindromic repeat), is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). Acts as a dsDNA endonuclease. Involved in the integration of spacer DNA into the CRISPR cassette.[HAMAP-Rule:MF_01470] [E6GPD6_ENTFL] CRISPR (clustered regularly interspaced short palindromic repeat), is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). Functions as a ssRNA-specific endoribonuclease. Involved in the integration of spacer DNA into the CRISPR cassette.[HAMAP-Rule:MF_01471] Publication Abstract from PubMedCRISPR (clustered regularly interspaced short palindromic repeats) and the nearby cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes1-5. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer6-9. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical7-9. The conserved Cas1 and Cas2 proteins form an integrase complex consisting two distal Cas1 dimers bridged by a Cas2 dimer in the middle6,10. The prespacer is bound by Cas1-Cas2 as a dual forked DNA, and the terminal 3'-OH of each 3'-overhang serves as an attacking nucleophile during integration11-14. Importantly, the prespacer is preferentially integrated into the leader-proximal region of the CRISPR array1,7,10,15, guided by the leader sequence and a pair of inverted repeats (IRs) inside the CRISPR repeat7,15-20. Spacer integration in the most well-studied Escherichia coli Type I-E CRISPR system further relies on the bacterial Integration Host Factor (IHF)21,22. In Type II-A CRISPR, however, Cas1-Cas2 alone integrates spacer efficiently in vitro18; other Cas proteins (Cas9 and Csn2) play accessory roles in prespacer biogenesis17,23. Focusing on the Enterococcus faecalis Type II-A system24, here we report four structure snapshots of Cas1-Cas2 during spacer integration. EfaCas1-Cas2 selectively binds to a splayed 30-bp prespacer bearing 4-nt 3'-overhangs. Three molecular events take place upon encountering a target: Cas1-Cas2/prespacer first searches for half-sites stochastically, then preferentially interacts with the leader-side CRISPR repeat and catalyzes a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3'-overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework explaining the stepwise spacer integration process and the leader-proximal preference. How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration.,Xiao Y, Ng S, Nam KH, Ke A Nature. 2017 Sep 4. doi: 10.1038/nature24020. PMID:28869593[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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