Characterization of the interaction between the restriction endonuclease McrBC from E. coli and its cofactor GTP

Pieper, Uwe; Brinkmann, Thilo; Krüger, Torsten; Noyer-Weidner, Mario; Pingoud, Alfred

doi:10.1006/jmbi.1997.1228

Cited by 29 publications

(52 citation statements)

References 32 publications

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“…It displays a conserved ENR signature and a further C-terminal acidic residue; the asparagine in the former motif is nearly absolutely conserved (Supplementary material). Studies on the McrBC system have indicated that McrC stimulates the intrinsic GTPase activity of McrB by 30-fold and is required to dimerize two McrB torroids 75, 79 . Based on this observation and the strict gene-neighborhood association, which we recovered between the genes for the McrB and McrC-NTD family proteins, we propose that the McrC-NTD specifically functions as stimulatory domain for the activity of the McrB NTPase.…”

Section: Resultsmentioning

confidence: 99%

Ter-dependent stress response systems: novel pathways related to metal sensing, production of a nucleoside-like metabolite, and DNA-processing

2012

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The mode of action of the bacterial ter cluster and TelA genes, implicated in natural resistance to tellurite and other xenobiotic toxic compounds, pore-forming colicins and several bacteriophages has remained enigmatic for almost two decades. Using comparative genomics, sequence-profile searches and structural analysis we present evidence that the ter gene products and their functional partners constitute previously underappreciated, chemical stress response and anti-viral defense systems of bacteria. Based on contextual information from conserved gene neighborhoods and domain architectures, we show that the ter gene products and TelA lie at the center of membrane-linked metal recognition complexes with regulatory ramifications encompassing phosphorylation-dependent signal transduction, RNA-dependent regulation, biosynthesis of nucleoside-like metabolites and DNA processing. Our analysis suggests that the multiple metal-binding and non-binding TerD paralogs and TerC are likely to constitute a membrane-associated complex, which might also include TerB and TerY, and feature several, distinct metal-binding sites. Versions of the TerB domain might also bind small molecule ligands and link the TerD paralog-TerC complex to biosynthetic modules comprised of phosphoribosyltransferases (PRTases), ATP grasp amidoligases, TIM-barrel carbon-carbon lyases, and HAD phosphoesterases, which are predicted to synthesize novel nucleoside-like molecules. One of the PRTases is also likely to interact with RNA by means of its Pelota/Ribosomal protein L7AE-like domain. The von Willebrand factor A domain protein, TerY, is predicted to be part of a distinct phosphorylation switch, coupling a protein kinase and a PP2C phosphatase. We show, based on the evidence from numerous conserved gene neighborhoods and domain architectures, that both the TerB and TelA domains have been linked to diverse lipid-interaction domains, such as two novel PH-like and the Coq4 domains, in different bacteria and are likely to comprise membrane-associated sensory complexes that might additionally contain periplasmic binding-protein-II and OmpA domains. The TerD and TerB domains and the TerY-associated phosphorylation system are also functionally linked to distinct DNA-processing complexes, which contain proteins with SWI2/SNF2 and RecQ-like helicases, multiple AAA+ ATPases, McrC-N-terminal domain proteins, several restriction endonuclease fold DNases, DNA-binding domains and a type-VII/Esx-like system, which is at the center of a predicted DNA transfer apparatus. These DNA-processing modules and associated genes are predicted to be involved in restriction or suicidal action in response to phages and possibly repairing xenobiotic-induced DNA damage. In some eukaryotes, certain components of the ter system appear to have recruited to function in conjunction with the ubiquitin system and calcium-signaling pathways.

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Section: Resultsmentioning

confidence: 99%

Ter-dependent stress response systems: novel pathways related to metal sensing, production of a nucleoside-like metabolite, and DNA-processing

2012

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“…These GTPases have been assigned to AAA+ class chaperonin-like ATPases [20] and include McrB of the E. coli methylation-dependent restriction system (McrBC). In this system, DNA cleavage by the McrC subunit is strictly coupled to GTP hydrolysis by the McrB subunit [21] instead of the typical ATP cofactor requirement of most restriction modification systems (for example Type I and Type III restriction endonucleases [22]. The McrC subunit responsible for cleavage is a PD-(D/E)XK endonuclease [21], which supports assignment of DUF524 to the restriction endonuclease-like superfamily and suggests a function of methylation-dependent restriction for this group of unknown proteins.…”

Section: Resultsmentioning

confidence: 99%

Realm of PD-(D/E)XK nuclease superfamily revisited: detection of novel families with modified transitive meta profile searches

et al. 2007

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Background: PD-(D/E)XK nucleases constitute a large and highly diverse superfamily of enzymes that display little sequence similarity despite retaining a common core fold and a few critical active site residues. This makes identification of new PD-(D/E)XK nuclease families a challenging task as they usually escape detection with standard sequence-based methods. We developed a modified transitive meta profile search approach and to consider the structural diversity of PD-(D/E)XK nuclease fold more thoroughly we analyzed also lower than threshold Meta-BASIC hits to select potentially correct predictions placed among unreliable or incorrect ones.

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“…It had been shown previously that MrcB is mainly responsible for GTP binding and cleavage (8,18,24), but also for specific DNA binding (13,19). This means that specific DNA binding and DNA cleavage are activities of two different subunits that have to cooperate to achieve specific DNA cleavage.…”

Section: Methodsmentioning

confidence: 95%

A Mutational Analysis of the PD...D/EXK Motif Suggests That McrC Harbors the Catalytic Center for DNA Cleavage by the GTP-Dependent Restriction Enzyme McrBC fromEscherichia coli

Pieper

Pingoud

2002

Biochemistry

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McrBC is a unique restriction enzyme which binds specifically to the bipartite recognition sequence R(m)CN( approximately )(30)(-)( approximately )(2000)R(m)C and in the presence of GTP translocates the DNA and cleaves both strands at multiple positions within the two R(m)C "half-sites". It is known that McrBC is composed of two subunits: McrB which binds and hydrolyzes GTP and specifically interacts with DNA and McrC whose function is not clear but which has been suspected to harbor the catalytic center for DNA cleavage. A multiple-sequence alignment of the amino acid sequence of Escherichia coli McrC and of six presumably homologous open reading frames from various bacterial species shows that a sequence motif found in many restriction enzymes, but also in other nucleases, the PD.D/EXK motif, is conserved among these sequences. A mutational analysis, in which the carboxylates (aspartic acid in McrC) of this motif were substituted with alanine or asparagine and lysine was substituted with alanine or arginine, strongly suggests that Asp244, Asp257, and Lys259 represent the catalytic center of E. coli McrC. Whereas the variants D244A (or -N), D257A (or -N), and K259A are inactive in DNA cleavage (K259R has residual DNA cleavage activity), they interact with McrB like wild-type McrC, as can be deduced from the finding that they stimulate the McrB-catalyzed GTP hydrolysis to the same extent as wild-type McrC. Thus, whereas McrC variants defective in DNA cleavage can stimulate the GTPase activity of McrB, the DNase activity of McrC is not supported by McrB variants defective in GTP hydrolysis.

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Characterization of the interaction between the restriction endonuclease McrBC from E. coli and its cofactor GTP

Cited by 29 publications

References 32 publications

Ter-dependent stress response systems: novel pathways related to metal sensing, production of a nucleoside-like metabolite, and DNA-processing

Ter-dependent stress response systems: novel pathways related to metal sensing, production of a nucleoside-like metabolite, and DNA-processing

Realm of PD-(D/E)XK nuclease superfamily revisited: detection of novel families with modified transitive meta profile searches

A Mutational Analysis of the PD...D/EXK Motif Suggests That McrC Harbors the Catalytic Center for DNA Cleavage by the GTP-Dependent Restriction Enzyme McrBC fromEscherichia coli

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