5h9f
From Proteopedia
Crystal structure of E. coli Cascade bound to a PAM-containing dsDNA target at 2.45 angstrom resolution.
Structural highlights
FunctionCSE1_ECOLI 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).[1] [2] [3] [4] A component of Cascade, which participates in CRISPR interference, the third stage of CRISPR immunity. Cascade binds both crRNA and in a sequence-specific manner negatively supercoiled dsDNA target. This leads to the formation of an R-loop in which the crRNA binds the target DNA, displacing the noncomplementary strand. Cas3 is recruited to Cascade, probably via interactions with CasA, nicks target DNA and then unwinds and cleaves the target, leading to DNA degradation and invader neutralization. CasA is not required for formation of Cascade, but probably enhances binding to and subsequent recognition of both target dsDNA and ssDNA.[5] [6] [7] [8] Publication Abstract from PubMedClustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 A crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 A apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process. Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli.,Hayes RP, Xiao Y, Ding F, van Erp PB, Rajashankar K, Bailey S, Wiedenheft B, Ke A Nature. 2016 Feb 25;530(7591):499-503. doi: 10.1038/nature16995. Epub 2016 Feb, 10. PMID:26863189[9] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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