Toroidal Structure and DNA Cleavage by the CRISPR-Associated [4Fe-4S] Cluster Containing Cas4 Nuclease SSO0001 from <i>Sulfolobus solfataricus</i>

Lemak, Sofia; Beloglazova, Natalia; Nocek, B.; Skarina, T.; Flick, Robert; Brown, Greg; Popović, Ana; Joachimiak, A.; Savchenko, Alexei; Yakunin, Alexander F.

doi:10.1021/ja408729b

Cited by 56 publications

(99 citation statements)

References 51 publications

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“…In contrast, the crystal structure of the Cas4 protein SSO0001 revealed a decameric toroid formed by five tightly bound dimers (19). Superimposition of structures of Pcal_0546 and SSO0001 reveals that, despite having similar overall architectures and active sites with a good overlap of the metal ions, the two structures differ considerably as shown by a large r.m.s.d.…”

Section: Resultsmentioning

confidence: 97%

“…The LC-MS analysis of the products of poly-dC 10 cleavage was performed essentially as described before (19) using a Q-Exactive mass spectrometer with HESI source and an Ulti-Mate 3000 UHPLC/autosampler system (all from Thermo Scientific). Separation by liquid chromatography was conducted on a Hypersil Gold C18 column (50 mm × 2.1 mm, 1.9 μm particle size, Thermo Scientific) equipped with a guard column.…”

Section: Methodsmentioning

confidence: 99%

“…Its crystal structure has revealed a toroidal decameric complex with each protomer containing one [4Fe-4S] cluster coordinated by the four conserved Cys residues (19). These cysteines comprise a signature motif of ‘iron staple’ nucleases, which have been proposed to use iron–sulfur clusters for the stabilization of the RecB-like nuclease domain (16,23).…”

Section: Introductionmentioning

confidence: 99%

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The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity

Lemak

Nocek

Beloglazova

et al. 2014

Nucleic Acids Research

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Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5′ to 3′ exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5′ to 3′ and 3′ to 5′ directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.

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Section: Resultsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity

Lemak

Nocek

Beloglazova

et al. 2014

Nucleic Acids Research

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“…Toroidal resonances in natural media remain to be observed and the spectroscopy of toroidal resonances to be developed. The similarity between the radiation properties of electric and toroidal dipoles calls for a re-examination of spectroscopic data, especially where systems of toroidal symmetry are involved, as is often the case with biological macromolecules 84 .…”

Section: Discussionmentioning

confidence: 99%

Electromagnetic toroidal excitations in matter and free space

et al. 2016

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The toroidal dipole is a localized electromagnetic excitation, distinct from the magnetic and electric dipoles. While the electric dipole can be understood as a pair of opposite charges and the magnetic dipole as a current loop, the toroidal dipole corresponds to currents flowing on the surface of a torus. Toroidal dipoles provide physically significant contributions to the basic characteristics of matter including absorption, dispersion, and optical activity. Toroidal excitations also exist in free-space as spatially and temporally localized electromagnetic pulses propagating at the speed of light and interacting with matter. We review recent experimental observations of resonant toroidal dipole excitations in metamaterials and the discovery of anapoles, non-radiating current configurations involving toroidal dipoles. While certain fundamental and practical aspects of toroidal electrodynamics remain open for the moment, we envision that exploitation of toroidal response can have important implications for the fields of photonics, sensing, energy and information. IntroductionThe interactions of electromagnetic radiation with matter underpin some of the most important technologies today -from telecommunications to information processing and data storage; from spectroscopy and imaging to light-assisted manufacturing. Our understanding and description of the electromagnetic properties of matter traditionally involves the concept of electric and magnetic dipoles, as well as their more complex combinations, known as multipoles. Introduced by Maxwell and Lorentz and later refined by Jackson and Landau, this framework, termed the multipole expansion, is central in physics and is being routinely applied in the study of optical, condensed matter, atomic, nuclear phenomena and beyond 1 . Within this framework, electromagnetic media can be represented by a set of point-like multipole sources [2][3][4][5] . The commonly used set of multipoles comprises the electric and magnetic families, which can be represented by oscillating charges and loop currents respectively. Dynamic toroidal multipoles constitute a third independent family of elementary electromagnetic sources, rather than an alternative multipole expansion or higher-order corrections to the conventional electric and magnetic multipoles (see tutorial inset I).Introduced in 1958 by Y. B. Zeldovich, toroidal moments have been considered in systems of toroidal topology (see Fig. 1) and studied in the context of nuclear 6 , atomic 7 , and molecular physics 8 , classical electrodynamics 9,10 , and solid state physics 3 . In the field of electromagnetism, in particular, a number of works have led to the development of a complete theoretical framework for toroidal electrodynamics [11][12][13][14][15] and the prediction of exotic effects, including dynamic non-radiating charge current configurations 10 and non-reciprocal interactions 9 . Following recent experimental observations of toroidal contributions in the response of materials across the electromagnetic spectrum, dyn...

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“…Although previously demonstrated as a ssDNA endonuclease (21), gp19 shares sequence similarity with different CRISPR-associated Cas4 proteins (24), which possess metal-dependent endonuclease and 5´→3´exonuclease activities against ssDNA (31). We therefore examined the…”

Section: Gp19 Demonstrates Both Ssdna Endonuclease and 5´-3´ Exonuclementioning

confidence: 99%

Functional Characterization of a Conserved Archaeal Viral Operon Revealing Single-Stranded DNA Binding, Annealing and Nuclease Activities

Guo

Kragelund

White

et al. 2015

Journal of Molecular Biology

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Toroidal Structure and DNA Cleavage by the CRISPR-Associated [4Fe-4S] Cluster Containing Cas4 Nuclease SSO0001 from Sulfolobus solfataricus

Cited by 56 publications

References 51 publications

The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity

The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity

Electromagnetic toroidal excitations in matter and free space

Functional Characterization of a Conserved Archaeal Viral Operon Revealing Single-Stranded DNA Binding, Annealing and Nuclease Activities

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