Dataset Information

Asymmetric positioning of Cas1-2 complex and Integration Host Factor induced DNA bending guide the unidirectional homing of protospacer in CRISPR-Cas type I-E system.

ABSTRACT: CRISPR-Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospacer invasion into the CRISPR array. However, the mechanism by which this protospacer fragment gets integrated in a directional fashion into the leader proximal end is elusive. Here, we employ CRISPR/dCas9 mediated immunoprecipitation and genetic analysis to identify Integration Host Factor (IHF) as an indispensable accessory factor for spacer acquisition in Escherichia coli Further, we show that the leader region abutting the first CRISPR repeat localizes IHF and Cas1-2 complex. IHF binding to the leader region induces bending by about 120° that in turn engenders the regeneration of the cognate binding site for protospacer bound Cas1-2 complex and brings it in proximity with the first CRISPR repeat. This appears to guide Cas1-2 complex to orient the protospacer invasion towards the leader-repeat junction thus driving the integration in a polarized fashion.

SUBMITTER: Yoganand KN

PROVIDER: S-EPMC5224486 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Asymmetric positioning of Cas1-2 complex and Integration Host Factor induced DNA bending guide the unidirectional homing of protospacer in CRISPR-Cas type I-E system.

Yoganand K N R KN Sivathanu R R Nimkar Siddharth S Anand B B

Nucleic acids research 20161129 1

CRISPR-Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospace ...[more]

PMID: 27899566

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Project description:CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical. The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer. 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 integration. The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array, guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat. Spacer integration in the well-studied Escherichia coli type I-E CRISPR system also relies on the bacterial integration host factor. In type II-A CRISPR, however, Cas1-Cas2 alone integrates spacers efficiently in vitro; other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers. Here we present four structural snapshots from the type II-A system of Enterococcus faecalis Cas1 and Cas2 during spacer integration. Enterococcus faecalis Cas1-Cas2 selectively binds to a splayed 30-base-pair prespacer bearing 4-nucleotide 3' overhangs. Three molecular events take place upon encountering a target: first, the Cas1-Cas2-prespacer complex searches for half-sites stochastically, then it preferentially interacts with the leader-side CRISPR repeat, and finally, it catalyses 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 to explain the stepwise spacer integration process and the leader-proximal preference.

Dataset Information

Asymmetric positioning of Cas1-2 complex and Integration Host Factor induced DNA bending guide the unidirectional homing of protospacer in CRISPR-Cas type I-E system.

Publications

Asymmetric positioning of Cas1-2 complex and Integration Host Factor induced DNA bending guide the unidirectional homing of protospacer in CRISPR-Cas type I-E system.

Similar Datasets

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