Mechanism and cleavage specificity of the H-N-H endonuclease colicin E9 1 1Edited by J. Karn

Pommer, Ansgar J.; Cal, Santiago; Keeble, Anthony H.; Walker, Daniel; Evans, Steven J.; Kühlmann, Ulrike C.; Cooper, Alan; Connolly, Bernard A.; Hemmings, Andrew M.; Moore, Geoffrey R.; James, Richard; Kleanthous, Colin

doi:10.1006/jmbi.2001.5189

Cited by 104 publications

(145 citation statements)

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“…Interestingly, the enzyme preferentially cleaves dsDNA after thymine, and consistent with this, a recent structure of the E9 DNase bound to dsDNA shows that it is bound to a scissile phosphodiester adjacent to a thymine base (448). Optimal endonucleolytic activity of the colicin E9 DNase against dsDNA substrates requires high concentrations (Ͼ5 mM) of either Mg 2ϩ or Ca 2ϩ and is optimal at alkaline pHs (Ͼ8), properties that are shared with many other H-N-H/ ␤␤␣-Me enzymes (524,526 (329,525), although in the case of the E9 DNase, these metals influence substrate specificity, with the enzyme preferentially cleaving single-stranded DNA in the presence of transition metal ions (524). Recent structures of the E9 DNase bound to dsDNA have highlighted the metal adaptability of the H-N-H/␤␤␣-Me motif, with structures of both Mg 2ϩ -and Zn 2ϩ -bound enzymes showing how the side chains of the motif rearrange to accommodate the octahedral and tetrahedral geometries, respectively, that each metal ion prefers (448).…”

Section: (I) Colicin Dnases (A) H-n-h/␤␤␣-me Enzymes By Another Namementioning

confidence: 59%

“…The E9 DNase has been shown to cleave both double-and single-stranded DNA substrates in vitro to yield 5Ј-phosphate and 3Ј-OH products with the enzyme most active against dsDNA (524). The enzyme will also cleave single-stranded RNA but poorly.…”

Section: (I) Colicin Dnases (A) H-n-h/␤␤␣-me Enzymes By Another Namementioning

confidence: 99%

“…Several observations cast serious doubt on zinc or indeed any other transition metal ion as the cofactor in vivo. First, the Kleanthous laboratory reported that zinc does not support E9 DNase activity and supports only very weak activity of the E7 DNase (524,525,639). The E9 DNase is much more active against supercoiled plasmids in the presence of Mg 2ϩ or Ca 2ϩ than transition metal ions (526).…”

Section: (I) Colicin Dnases (A) H-n-h/␤␤␣-me Enzymes By Another Namementioning

confidence: 99%

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Colicin Biology

Cascales

Buchanan

Duché

et al. 2007

Microbiol Mol Biol Rev

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984

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SUMMARY Colicins are proteins produced by and toxic for some strains of Escherichia coli. They are produced by strains of E. coli carrying a colicinogenic plasmid that bears the genetic determinants for colicin synthesis, immunity, and release. Insights gained into each fundamental aspect of their biology are presented: their synthesis, which is under SOS regulation; their release into the extracellular medium, which involves the colicin lysis protein; and their uptake mechanisms and modes of action. Colicins are organized into three domains, each one involved in a different step of the process of killing sensitive bacteria. The structures of some colicins are known at the atomic level and are discussed. Colicins exert their lethal action by first binding to specific receptors, which are outer membrane proteins used for the entry of specific nutrients. They are then translocated through the outer membrane and transit through the periplasm by either the Tol or the TonB system. The components of each system are known, and their implication in the functioning of the system is described. Colicins then reach their lethal target and act either by forming a voltage-dependent channel into the inner membrane or by using their endonuclease activity on DNA, rRNA, or tRNA. The mechanisms of inhibition by specific and cognate immunity proteins are presented. Finally, the use of colicins as laboratory or biotechnological tools and their mode of evolution are discussed.

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Section: (I) Colicin Dnases (A) H-n-h/␤␤␣-me Enzymes By Another Namementioning

confidence: 59%

Section: (I) Colicin Dnases (A) H-n-h/␤␤␣-me Enzymes By Another Namementioning

confidence: 99%

Section: (I) Colicin Dnases (A) H-n-h/␤␤␣-me Enzymes By Another Namementioning

confidence: 99%

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Colicin Biology

Cascales

Buchanan

Duché

et al. 2007

Microbiol Mol Biol Rev

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984

1,359

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“…The E9 DNase binds Zn 2ϩ ions with nM affinity, and this interaction considerably stabilizes the protein (19). However, for the DNase domain to be enzymatically active in vivo, Mg 2ϩ ions are required, although these do not bind directly to the protein in the absence of DNA (16,20). Intoxication of E. coli cells by colicin E9 induces the SOS response, the characteristic response to DNA damage, prior to cell death (21).…”

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confidence: 99%

Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids

Mosbahi

Walker

Lea

et al. 2004

Journal of Biological Chemistry

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We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers (Mosbahi, K., Lemaître, C., Keeble, A. H., Mobasheri, H., Morel, B., James, R., Moore, G. R., Lea, E. J., and Kleanthous, C. (2002) Nat. Struct. Biol. 9, 476 -484). It was proposed that the E9 DNase mediates its own translocation across the cytoplasmic membrane and that the formation of ion channels is essential to this process. Here we describe changes to the structure and stability of the E9 DNase that accompany interaction of the protein with phospholipid vesicles. Formation of the protein-lipid complex at pH 7.5 resulted in a red-shift of the intrinsic protein fluorescence emission maximum ( max ) from 333 to 346 nm. At pH 4.0, where the E9 DNase lacks tertiary structure but retains secondary structure, DOPG induced a blue-shift in max , from 354 to 342 nm. Changes in max were specific for anionic phospholipid vesicles at both pHs, suggesting electrostatics play a role in this association. The effects of phospholipid were negated by Im9 binding, the high affinity, acidic, exosite inhibitor protein, but not by zinc, which binds at the active site. Fluorescence-quenching experiments further demonstrated that similar protein-phospholipid complexes are formed regardless of whether the E9 DNase is initially in its native conformation. Consistent with these observations, chemical and thermal denaturation data as well as proteolytic susceptibility experiments showed that association with negatively charged phospholipids destabilize the E9 DNase. We suggest that formation of a destabilizing protein-lipid complex preempts channel formation by the E9 DNase and constitutes the initial step in its translocation across the Escherichia coli inner membrane.Unraveling the interactions and mechanisms that enable proteins to cross biological membranes is of considerable interest, as the ability to target specific exogenous enzymes to the cytosol is likely to facilitate the design and discovery of novel chemotherapeutic agents. Many protein toxins have evolved to deliver a cytotoxic domain or subunit to the cytoplasm of susceptible cells, and so they provide an invaluable tool for studying protein translocation from the extracellular environment to their cellular targets, often located in the cytoplasm (1). The transition from the water-soluble to membrane-bound state has perhaps been most intensely studied in the pore-forming colicins (2, 3). This family of bacterial toxins, like all colicins, share a common three-domain structure, with receptor-binding and translocation domains that facilitate binding to the cell surface and mediate delivery of the channel-forming cytotoxic domain to the inner membrane. Cell death occurs as a consequence of ion channel formation across the cytoplasmic membrane, inducing depolarization of the membrane. Association of the cytotoxic domain with the membrane is thought to lead to destabilization and unfolding of the protein, yielding a "molten...

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“…Interestingly, although HNH domains show high similarities, the enzymes are not activated by the same metal ions (8,9,14,22,32,33). Several metal ions have been reported to stabilize the protein or are necessary for either DNA binding or cleavage.…”

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confidence: 99%

Bacillus subtilis hlpB Encodes a Conserved Stand-Alone HNH Nuclease-Like Protein That Is Essential for Viability Unless the hlpB Deletion Is Accompanied by the Deletion of Genes Encoding the AddAB DNA Repair Complex

2012

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bThe HNH domain is found in many different proteins in all phylogenetic kingdoms and in many cases confers nuclease activity. We have found that the Bacillus subtilis hlpB (yisB) gene encodes a stand-alone HNH domain, homologs of which are present in several bacterial genomes. We show that the protein we term HlpB is essential for viability. The depletion of HlpB leads to growth arrest and to the generation of cells containing a single, decondensed nucleoid. This apparent condensation-segregation defect was cured by additional hlpB copies in trans. Purified HlpB showed cooperative binding to a variety of double-stranded and single-stranded DNA sequences, depending on the presence of zinc, nickel, or cobalt ions. Binding of HlpB was also influenced by pH and different metals, reminiscent of HNH domains. Lethality of the hlpB deletion was relieved in the absence of addA and of addAB, two genes encoding proteins forming a RecBCD-like end resection complex, but not of recJ, which is responsible for a second end-resectioning avenue. Like AddA-green fluorescent protein (AddA-GFP), functional HlpB-YFP or HlpB-FlAsH fusions were present throughout the cytosol in growing B. subtilis cells. Upon induction of DNA damage, HlpBFlAsH formed a single focus on the nucleoid in a subset of cells, many of which colocalized with the replication machinery. Our data suggest that HlpB plays a role in DNA repair by rescuing AddAB-mediated recombination intermediates in B. subtilis and possibly also in many other bacteria. HNH domains exist in a broad spectrum of different organisms such as bacteria, phages, viruses, archaea, and eukaryotes (7). To date, more than 2,000 proteins containing an HNH motif have been identified. HNH stands for the three most conserved histidine and asparagine residues located within a nucleic acid-binding and cleavage site, which is composed of 30 to 40 amino acid (aa) residues (15). The HNH motif is found in a variety of different enzymes, including homing endonucleases, which initiate the insertion of mobile genetic elements into specific sites (such as selfsplicing introns and inteins, e.g., yeast intron 1 protein), MutS homologs involved in DNA mismatch repair, bacterial toxins such as colicins and pyocins that degrade chromosomal DNA or rRNA (7,23,26), restriction endonucleases, e.g., methyl cytosine-specific restriction endonuclease MrcA of Escherichia coli (2), transposases, and DNA packaging factors (6). Immunity proteins are coexpressed with and tightly bound to colicins to avoid degradation of host DNA (18). In addition, HNH domains are found in proteins such as reverse transcriptases (34) and DNases. All these proteins play important roles in many different cellular processes, e.g., DNA repair, replication, and recombination and processes related to RNA (8).HNH domains are best characterized in colicins, where they clearly provide nuclease activity. The first crystal structures of HNH domains were resolved in the nuclease domains of E. coli colicins E7 and E9 (4,18,19). The structures revealed tha...

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Mechanism and cleavage specificity of the H-N-H endonuclease colicin E9 1 1Edited by J. Karn

Cited by 104 publications

References 46 publications

Colicin Biology

Colicin Biology

Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids

Bacillus subtilis hlpB Encodes a Conserved Stand-Alone HNH Nuclease-Like Protein That Is Essential for Viability Unless the hlpB Deletion Is Accompanied by the Deletion of Genes Encoding the AddAB DNA Repair Complex

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