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CRISPR type II-A subgroups exhibit phylogenetically distinct mechanisms for prespacer insertion.

ABSTRACT: CRISPR-Cas is an adaptive immune system that protects prokaryotes against foreign nucleic acids. Prokaryotes gain immunity by acquiring short pieces of the invading nucleic acid termed prespacers and inserting them into their CRISPR array. In type II-A systems, Cas1 and Cas2 proteins insert prespacers always at the leader-repeat junction of the CRISPR array. Among type II-A CRISPR systems, three distinct groups (G1, G2, and G3) exist according to the extent of DNA sequence conservation at the 3' end of the leader. However, the mechanisms by which these conserved motifs interact with their cognate Cas1 and Cas2 proteins remain unclear. Here, we performed in vitro integration assays, finding that for G1 and G2, the insertion site is recognized through defined mechanisms, at least in members examined to date, whereas G3 exhibits no sequence-specific insertion. G1 first recognized a 12-bp sequence at the leader-repeat junction and performed leader-side insertion before proceeding to spacer-side insertion. G2 recognized the full repeat sequence and could perform independent leader-side or spacer-side insertions, although the leader-side insertion was faster than spacer-side. The prespacer morphology requirements for Cas1-Cas2 varied, with G1 stringently requiring a 5-nucleotide 3' overhang and G2 being able to insert many forms of prespacers with variable efficiencies. These results highlight the intricacy of protein-DNA sequence interactions within the seemingly similar type II-A integration complexes and provide mechanistic insights into prespacer insertion. These interactions can be fine-tuned to expand the Cas1-Cas2 toolset for inserting small DNAs into diverse DNA targets.

SUBMITTER: Van Orden MJ

PROVIDER: S-EPMC7415960 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSeven transmembrane receptors (7TMRs) can adopt different active conformations facilitating a selective activation of either G protein or ?-arrestin-dependent signaling pathways. This represents an opportunity for development of novel therapeutics targeting selective biological effects of a given receptor. Several studies on pathway separation have been performed, many of these on the Angiotensin II type 1 receptor (AT1R). It has been shown that certain ligands or mutations facilitate internalization and/or recruitment of ?-arrestins without activation of G proteins. However, the underlying molecular mechanisms remain largely unresolved. For instance, it is unclear whether such selective G protein-uncoupling is caused by a lack of ability to interact with G proteins or rather by an increased ability of the receptor to recruit ?-arrestins. Since uncoupling of G proteins by increased ability to recruit ?-arrestins could lead to different cellular or in vivo outcomes than lack of ability to interact with G proteins, it is essential to distinguish between these two mechanisms.Methodology/principal findingsWe studied five AT1R mutants previously published to display pathway separation: D74N, DRY/AAY, Y292F, N298A, and Y302F (Ballesteros-Weinstein numbering: 2.50, 3.49-3.51, 7.43, 7.49, and 7.53). We find that D74N, DRY/AAY, and N298A mutants are more prone to ?-arrestin recruitment than WT. In contrast, receptor mutants Y292F and Y302F showed impaired ability to recruit ?-arrestin in response to Sar1-Ile4-Ile8 (SII) Ang II, a ligand solely activating the ?-arrestin pathway.Conclusions/significanceOur analysis reveals that the underlying conformations induced by these AT1R mutants most likely represent principally different mechanisms of uncoupling the G protein, which for some mutants may be due to their increased ability to recruit ?-arrestin2. Hereby, these findings have important implications for drug discovery and 7TMR biology and illustrate the necessity of uncovering the exact molecular determinants for G protein-coupling and ?-arrestin recruitment, respectively.

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CRISPR type II-A subgroups exhibit phylogenetically distinct mechanisms for prespacer insertion.

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