Dataset Information

Probing the Behaviour of Cas1-Cas2 upon Protospacer Binding in CRISPR-Cas Systems using Molecular Dynamics Simulations.

ABSTRACT: Adaptation in CRISPR-Cas systems enables the generation of an immunological memory to defend against invading viruses. This process is driven by foreign DNA spacer (termed protospacer) selection and integration mediated by Cas1-Cas2 protein. Recently, different states of Cas1-Cas2, in its free form and in complex with protospacer DNAs, were solved by X-ray crystallography. In this paper, molecular dynamics (MD) simulations are employed to study crystal structures of one free and two protospacer-bound Cas1-Cas2 complexes. The simulated results indicate that the protospacer binding markedly increases the system stability, in particular when the protospacer containing the PAM-complementary sequence. The hydrogen bond and binding free energy calculations explain that PAM recognition introduces more specific interactions to increase the cleavage activity of Cas1. By using principal component analysis (PCA) and intramolecular angle calculation, this study observes two dominant slow motions associated with the binding of Ca1-Cas2 to the protospacer and potential target DNAs respectively. The comparison of DNA structural deformation further implies a cooperative conformational change of Cas1-Cas2 and protospacer for the target DNA capture. We propose that this cooperativity is the intrinsic requirement of the CRISPR integration complex formation. This study provides some new insights into the understanding of CRISPR-Cas adaptation.

SUBMITTER: Wan H

PROVIDER: S-EPMC6395717 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Probing the Behaviour of Cas1-Cas2 upon Protospacer Binding in CRISPR-Cas Systems using Molecular Dynamics Simulations.

Wan Hua H Li Jianming J Chang Shan S Lin Shuoxin S Tian Yuanxin Y Tian Xuhong X Wang Meihua M Hu Jianping J

Scientific reports 20190228 1

Adaptation in CRISPR-Cas systems enables the generation of an immunological memory to defend against invading viruses. This process is driven by foreign DNA spacer (termed protospacer) selection and integration mediated by Cas1-Cas2 protein. Recently, different states of Cas1-Cas2, in its free form and in complex with protospacer DNAs, were solved by X-ray crystallography. In this paper, molecular dynamics (MD) simulations are employed to study crystal structures of one free and two protospacer- ...[more]

PMID: 30816277

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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

Probing the Behaviour of Cas1-Cas2 upon Protospacer Binding in CRISPR-Cas Systems using Molecular Dynamics Simulations.

Publications

Probing the Behaviour of Cas1-Cas2 upon Protospacer Binding in CRISPR-Cas Systems using Molecular Dynamics Simulations.

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