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Mechanism of spacer integration links the CRISPR/Cas system to transposition as a form of mobile DNA.

Dyda F, Hickman AB - Mob DNA (2015)

Bottom Line: These are typified by multiple copies of DNA sequences known as clustered regularly interspaced short palindromic repeats (CRISPRs).These CRISPR repeats are the sites at which short spacers containing sequences of previously encountered foreign DNA are integrated, and the spacers serve as the molecular memory of previous invaders.In vivo work has demonstrated that two CRISPR-associated proteins - Cas1 and Cas2 - are required for spacer integration, but the mechanism by which this is accomplished remained unclear.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 5 Center Dr., Bethesda, MD 20892 USA.

ABSTRACT
It has recently become clear that many bacterial and archaeal species possess adaptive immune systems. These are typified by multiple copies of DNA sequences known as clustered regularly interspaced short palindromic repeats (CRISPRs). These CRISPR repeats are the sites at which short spacers containing sequences of previously encountered foreign DNA are integrated, and the spacers serve as the molecular memory of previous invaders. In vivo work has demonstrated that two CRISPR-associated proteins - Cas1 and Cas2 - are required for spacer integration, but the mechanism by which this is accomplished remained unclear. Here we review a recent paper describing the in vitro reconstitution of CRISPR spacer integration using purified Cas1 and Cas2 and place the results in context of similar DNA transposition reactions and the crystal structure of the Cas1/Cas2 complex.

No MeSH data available.


Related in: MedlinePlus

Structure of the Cas1/Cas2 complex (PDB code 4P6I). Cas1 protomers (labelled as in [7]) are shown in gold and orange, the central Cas2 dimer in blue and green. (A) Arrows show the active sites (marked by His208, shown as blue sticks) of the two Cas1 protomers that bind directly to the Cas2 dimer. The distance between the active sites, which point away from each other, is approximately 81 Å. (B) Horizontal rotation of the view of the complex assembly reveals that the active sites of the other two Cas1 protomers in the heterohexamer are more convincingly pointed towards each other (as expected if these active sites mediate coordinated integration of a protospacer) and are separated by approximately 93 Å. However, the as-the-crow-flies connection between these two active sites passes directly through the intervening Cas2 dimer.
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Fig2: Structure of the Cas1/Cas2 complex (PDB code 4P6I). Cas1 protomers (labelled as in [7]) are shown in gold and orange, the central Cas2 dimer in blue and green. (A) Arrows show the active sites (marked by His208, shown as blue sticks) of the two Cas1 protomers that bind directly to the Cas2 dimer. The distance between the active sites, which point away from each other, is approximately 81 Å. (B) Horizontal rotation of the view of the complex assembly reveals that the active sites of the other two Cas1 protomers in the heterohexamer are more convincingly pointed towards each other (as expected if these active sites mediate coordinated integration of a protospacer) and are separated by approximately 93 Å. However, the as-the-crow-flies connection between these two active sites passes directly through the intervening Cas2 dimer.

Mentions: Taken together, the features of the reconstituted in vitro system established in [3] are consistent with in vivo data and display strong similarities to DNA transposition. However, the protein architectures of neither Cas1 nor Cas2 conform with the paradigms of those known DNA transposases that use direct nucleophilic attack, as they are not members of the retroviral integrase superfamily and do not use three carboxylate side chains (the so-called DDE/D motif) as active site residues. Rather, Cas1 has a unique fold and an active site residue constellation of conserved E, N, H, and D/E residues [6] (Figure 2A). The E/H/D triad has been observed to coordinate a divalent metal ion [6], and mutation of the D abolished the nonspecific nuclease activity of Cas1 [6]. Reassuringly, mutations of the metal-coordinating H and D residues in Cas1 abolished protospacer integration in vitro, implicating the involvement of Cas1’s active site.Figure 2


Mechanism of spacer integration links the CRISPR/Cas system to transposition as a form of mobile DNA.

Dyda F, Hickman AB - Mob DNA (2015)

Structure of the Cas1/Cas2 complex (PDB code 4P6I). Cas1 protomers (labelled as in [7]) are shown in gold and orange, the central Cas2 dimer in blue and green. (A) Arrows show the active sites (marked by His208, shown as blue sticks) of the two Cas1 protomers that bind directly to the Cas2 dimer. The distance between the active sites, which point away from each other, is approximately 81 Å. (B) Horizontal rotation of the view of the complex assembly reveals that the active sites of the other two Cas1 protomers in the heterohexamer are more convincingly pointed towards each other (as expected if these active sites mediate coordinated integration of a protospacer) and are separated by approximately 93 Å. However, the as-the-crow-flies connection between these two active sites passes directly through the intervening Cas2 dimer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940900&req=5

Fig2: Structure of the Cas1/Cas2 complex (PDB code 4P6I). Cas1 protomers (labelled as in [7]) are shown in gold and orange, the central Cas2 dimer in blue and green. (A) Arrows show the active sites (marked by His208, shown as blue sticks) of the two Cas1 protomers that bind directly to the Cas2 dimer. The distance between the active sites, which point away from each other, is approximately 81 Å. (B) Horizontal rotation of the view of the complex assembly reveals that the active sites of the other two Cas1 protomers in the heterohexamer are more convincingly pointed towards each other (as expected if these active sites mediate coordinated integration of a protospacer) and are separated by approximately 93 Å. However, the as-the-crow-flies connection between these two active sites passes directly through the intervening Cas2 dimer.
Mentions: Taken together, the features of the reconstituted in vitro system established in [3] are consistent with in vivo data and display strong similarities to DNA transposition. However, the protein architectures of neither Cas1 nor Cas2 conform with the paradigms of those known DNA transposases that use direct nucleophilic attack, as they are not members of the retroviral integrase superfamily and do not use three carboxylate side chains (the so-called DDE/D motif) as active site residues. Rather, Cas1 has a unique fold and an active site residue constellation of conserved E, N, H, and D/E residues [6] (Figure 2A). The E/H/D triad has been observed to coordinate a divalent metal ion [6], and mutation of the D abolished the nonspecific nuclease activity of Cas1 [6]. Reassuringly, mutations of the metal-coordinating H and D residues in Cas1 abolished protospacer integration in vitro, implicating the involvement of Cas1’s active site.Figure 2

Bottom Line: These are typified by multiple copies of DNA sequences known as clustered regularly interspaced short palindromic repeats (CRISPRs).These CRISPR repeats are the sites at which short spacers containing sequences of previously encountered foreign DNA are integrated, and the spacers serve as the molecular memory of previous invaders.In vivo work has demonstrated that two CRISPR-associated proteins - Cas1 and Cas2 - are required for spacer integration, but the mechanism by which this is accomplished remained unclear.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 5 Center Dr., Bethesda, MD 20892 USA.

ABSTRACT
It has recently become clear that many bacterial and archaeal species possess adaptive immune systems. These are typified by multiple copies of DNA sequences known as clustered regularly interspaced short palindromic repeats (CRISPRs). These CRISPR repeats are the sites at which short spacers containing sequences of previously encountered foreign DNA are integrated, and the spacers serve as the molecular memory of previous invaders. In vivo work has demonstrated that two CRISPR-associated proteins - Cas1 and Cas2 - are required for spacer integration, but the mechanism by which this is accomplished remained unclear. Here we review a recent paper describing the in vitro reconstitution of CRISPR spacer integration using purified Cas1 and Cas2 and place the results in context of similar DNA transposition reactions and the crystal structure of the Cas1/Cas2 complex.

No MeSH data available.


Related in: MedlinePlus