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1.  Mechanosensitive channels and bacterial cell wall integrity: does life end with a bang or a whimper? 
Mechanogated channels are fundamental components of bacterial cells that enable retention of physical integrity during extreme increases in cell turgor. Optical tweezers combined with microfluidics have been used to study the fate of individual Escherichia coli cells lacking such channels when subjected to a bursting stress caused by increased turgor. Fluorescence-activated cell sorting and electron microscopy complement these studies. These analyses show that lysis occurs with a high probability, but the precise path differs between individual cells. By monitoring the loss of cytoplasmic green fluorescent protein, we have determined that some cells release this protein but remain phase dark (granular) consistent with the retention of the majority of large proteins. By contrast, most cells suffer cataclysmic wall failure leading to loss of granularity but with the retention of DNA and overall cell shape (protein-depleted ghosts). The time span of these events induced by hypo-osmotic shock varies but is of the order of milliseconds. The data are interpreted in terms of the timing of mechanosensitive channel gating relative to osmotically induced water influx.
doi:10.1098/rsif.2013.0850
PMCID: PMC3869158  PMID: 24258154
mechanosensitive channels; (bacterial) cell wall; bacterial stress response; optical tweezers; microfluidics; fluorescence-activated cell sorting
2.  ArdA proteins from different mobile genetic elements can bind to the EcoKI Type I DNA methyltransferase of E. coli K12☆ 
Biochimica et Biophysica Acta  2014;1844(3):505-511.
Anti-restriction and anti-modification (anti-RM) is the ability to prevent cleavage by DNA restriction–modification (RM) systems of foreign DNA entering a new bacterial host. The evolutionary consequence of anti-RM is the enhanced dissemination of mobile genetic elements. Homologues of ArdA anti-RM proteins are encoded by genes present in many mobile genetic elements such as conjugative plasmids and transposons within bacterial genomes. The ArdA proteins cause anti-RM by mimicking the DNA structure bound by Type I RM enzymes. We have investigated ArdA proteins from the genomes of Enterococcus faecalis V583, Staphylococcus aureus Mu50 and Bacteroides fragilis NCTC 9343, and compared them to the ArdA protein expressed by the conjugative transposon Tn916. We find that despite having very different structural stability and secondary structure content, they can all bind to the EcoKI methyltransferase, a core component of the EcoKI Type I RM system. This finding indicates that the less structured ArdA proteins become fully folded upon binding. The ability of ArdA from diverse mobile elements to inhibit Type I RM systems from other bacteria suggests that they are an advantage for transfer not only between closely-related bacteria but also between more distantly related bacterial species.
Highlights
•Diverse ArdA proteins all target the EcoKI Type I DNA modification enzyme.•ArdA proteins have variable secondary structure content.•ArdA all bind equally well to EcoKI despite stability variations.
doi:10.1016/j.bbapap.2013.12.008
PMCID: PMC3969726  PMID: 24368349
RM, restriction–modification; anti-RM, antirestriction/antimodification; MGE, mobile genetic element; MTase, modification methyltransferase; M subunit, modification subunit; S subunit, sequence specificity subunit; Orf, open reading frame; CD, circular dichroism; GuCl, guanidinium chloride; 2-ME, 2-mercaptoethanol; SEC, size exclusion chromatography; Kd, dissociation constant; DNA methyltransferase; ArdA protein; DNA mimic; Horizontal gene transfer
3.  Highlights of the DNA cutters: a short history of the restriction enzymes 
Nucleic Acids Research  2013;42(1):3-19.
In the early 1950’s, ‘host-controlled variation in bacterial viruses’ was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.
doi:10.1093/nar/gkt990
PMCID: PMC3874209  PMID: 24141096
4.  Type I restriction enzymes and their relatives 
Nucleic Acids Research  2013;42(1):20-44.
Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction–modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes.
doi:10.1093/nar/gkt847
PMCID: PMC3874165  PMID: 24068554
5.  Type III restriction-modification enzymes: a historical perspective 
Nucleic Acids Research  2013;42(1):45-55.
Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction–modification (R–M) systems are classified into four groups. Type III R–M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25–27 bp downstream of one of the recognition sites). Like the Type I R–M enzymes, Type III R–M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R–M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R–M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.
doi:10.1093/nar/gkt616
PMCID: PMC3874151  PMID: 23863841
6.  Removal of a frameshift between the hsdM and hsdS genes of the EcoKI Type IA DNA restriction and modification system produces a new type of system and links the different families of Type I systems 
Nucleic Acids Research  2012;40(21):10916-10924.
The EcoKI DNA methyltransferase is a trimeric protein comprised of two modification subunits (M) and one sequence specificity subunit (S). This enzyme forms the core of the EcoKI restriction/modification (RM) enzyme. The 3′ end of the gene encoding the M subunit overlaps by 1 nt the start of the gene for the S subunit. Translation from the two different open reading frames is translationally coupled. Mutagenesis to remove the frameshift and fuse the two subunits together produces a functional RM enzyme in vivo with the same properties as the natural EcoKI system. The fusion protein can be purified and forms an active restriction enzyme upon addition of restriction subunits and of additional M subunit. The Type I RM systems are grouped into families, IA to IE, defined by complementation, hybridization and sequence similarity. The fusion protein forms an evolutionary intermediate form lying between the Type IA family of RM enzymes and the Type IB family of RM enzymes which have the frameshift located at a different part of the gene sequence.
doi:10.1093/nar/gks876
PMCID: PMC3510504  PMID: 23002145
7.  Impact of target site distribution for Type I restriction enzymes on the evolution of methicillin-resistant Staphylococcus aureus (MRSA) populations 
Nucleic Acids Research  2013;41(15):7472-7484.
A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants. We have identified the specific DNA sequence targets for the dominant MRSA lineages CC1, CC5, CC8 and ST239. We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently. Target sites are distributed randomly in S. aureus genomes, except in a set of large conjugative plasmids encoding resistance genes that show evidence of spreading between two successful MRSA lineages. This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium. We show that a lack of specific target sites enables plasmids to evade the Type I RM system thereby contributing to the evolution of increasingly resistant community and hospital MRSA.
doi:10.1093/nar/gkt535
PMCID: PMC3753647  PMID: 23771140
8.  Exploring the DNA mimicry of the Ocr protein of phage T7 
Nucleic Acids Research  2012;40(16):8129-8143.
DNA mimic proteins have evolved to control DNA-binding proteins by competing with the target DNA for binding to the protein. The Ocr protein of bacteriophage T7 is the most studied DNA mimic and functions to block the DNA-binding groove of Type I DNA restriction/modification enzymes. This binding prevents the enzyme from cleaving invading phage DNA. Each 116 amino acid monomer of the Ocr dimer has an unusual amino acid composition with 34 negatively charged side chains but only 6 positively charged side chains. Extensive mutagenesis of the charges of Ocr revealed a regression of Ocr activity from wild-type activity to partial activity then to variants inactive in antirestriction but deleterious for cell viability and lastly to totally inactive variants with no deleterious effect on cell viability. Throughout the mutagenesis the Ocr mutant proteins retained their folding. Our results show that the extreme bias in charged amino acids is not necessary for antirestriction activity but that less charged variants can affect cell viability by leading to restriction proficient but modification deficient cell phenotypes.
doi:10.1093/nar/gks516
PMCID: PMC3439906  PMID: 22684506
9.  A unique homologue of the eukaryotic protein-modifier ubiquitin present in the bacterium Bacteroides fragilis, a predominant resident of the human gastrointestinal tract 
Microbiology  2011;157(Pt 11):3071-3078.
In the complete genome sequences of Bacteroides fragilis NCTC9343 and 638R, we have discovered a gene, ubb, the product of which has 63 % identity to human ubiquitin and cross-reacts with antibodies raised against bovine ubiquitin. The sequence of ubb is closest in identity (76 %) to the ubiquitin gene from a migratory grasshopper entomopoxvirus, suggesting acquisition by inter-kingdom horizontal gene transfer. We have screened clinical isolates of B. fragilis from diverse geographical regions and found that ubb is present in some, but not all, strains. The gene is transcribed and the mRNA is translated in B. fragilis, but deletion of ubb did not have a detrimental effect on growth. BfUbb has a predicted signal sequence; both full-length and processed forms were detected in whole-cell extracts, while the processed form was found in concentrated culture supernatants. Purified recombinant BfUbb inhibited in vitro ubiquitination and was able to covalently bind the human E1 activating enzyme, suggesting it could act as a suicide substrate in vivo. B. fragilis is one of the predominant members of the normal human gastrointestinal microbiota with estimates of up to >1011 cells per g faeces by culture. These data indicate that the gastro-intestinal tract of some individuals could contain a significant amount of aberrant ubiquitin with the potential to inappropriately activate the host immune system and/or interfere with eukaryotic ubiquitin activity. This discovery could have profound implications in relation to our understanding of human diseases such as inflammatory bowel and autoimmune diseases.
doi:10.1099/mic.0.049940-0
PMCID: PMC3352274  PMID: 21885481
10.  An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme 
Nucleic Acids Research  2011;39(17):7667-7676.
Type I DNA restriction/modification systems are oligomeric enzymes capable of switching between a methyltransferase function on hemimethylated host DNA and an endonuclease function on unmethylated foreign DNA. They have long been believed to not turnover as endonucleases with the enzyme becoming inactive after cleavage. Cleavage is preceded and followed by extensive ATP hydrolysis and DNA translocation. A role for dissociation of subunits to allow their reuse has been proposed for the EcoR124I enzyme. The EcoKI enzyme is a stable assembly in the absence of DNA, so recycling was thought impossible. Here, we demonstrate that EcoKI becomes unstable on long unmethylated DNA; reuse of the methyltransferase subunits is possible so that restriction proceeds until the restriction subunits have been depleted. We observed that RecBCD exonuclease halts restriction and does not assist recycling. We examined the DNA structure required to initiate ATP hydrolysis by EcoKI and find that a 21-bp duplex with single-stranded extensions of 12 bases on either side of the target sequence is sufficient to support hydrolysis. Lastly, we discuss whether turnover is an evolutionary requirement for restriction, show that the ATP hydrolysis is not deleterious to the host cell and discuss how foreign DNA occasionally becomes fully methylated by these systems.
doi:10.1093/nar/gkr480
PMCID: PMC3177214  PMID: 21685455
11.  DNA translocation by type III restriction enzymes: a comparison of current models of their operation derived from ensemble and single-molecule measurements 
Nucleic Acids Research  2011;39(11):4525-4531.
Much insight into the interactions of DNA and enzymes has been obtained using a number of single-molecule techniques. However, recent results generated using two of these techniques—atomic force microscopy (AFM) and magnetic tweezers (MT)—have produced apparently contradictory results when applied to the action of the ATP-dependent type III restriction endonucleases on DNA. The AFM images show extensive looping of the DNA brought about by the existence of multiple DNA binding sites on each enzyme and enzyme dimerisation. The MT experiments show no evidence for looping being a requirement for DNA cleavage, but instead support a diffusive sliding of the enzyme on the DNA until an enzyme–enzyme collision occurs, leading to cleavage. Not only do these two methods appear to disagree, but also the models derived from them have difficulty explaining some ensemble biochemical results on DNA cleavage. In this ‘Survey and Summary’, we describe several different models put forward for the action of type III restriction enzymes and their inadequacies. We also attempt to reconcile the different models and indicate areas for further experimentation to elucidate the mechanism of these enzymes.
doi:10.1093/nar/gkq1285
PMCID: PMC3113558  PMID: 21310716
12.  Single-molecule imaging of Bacteroides fragilis AddAB reveals the highly processive translocation of a single motor helicase 
Nucleic Acids Research  2010;38(11):3721-3731.
The AddAB helicase and nuclease complex is used for repairing double-strand DNA breaks in the many bacteria that do not possess RecBCD. Here, we show that AddAB, from the Gram-negative opportunistic pathogen Bacteroides fragilis, can rescue the ultraviolet sensitivity of an Escherichia coli recBCD mutant and that addAB is required for survival of B. fragilis following DNA damage. Using single-molecule observations we demonstrate that AddAB can translocate along DNA at up to 250 bp per second and can unwind an average of 14 000 bp, with some complexes capable of unwinding 40 000 bp. These results demonstrate the importance of processivity for facilitating encounters with recognition sequences that modify enzyme function during homologous recombination.
doi:10.1093/nar/gkq100
PMCID: PMC2887965  PMID: 20185564
13.  The structure of the KlcA and ArdB proteins reveals a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro 
Nucleic Acids Research  2009;38(5):1723-1737.
Plasmids, conjugative transposons and phage frequently encode anti-restriction proteins to enhance their chances of entering a new bacterial host that is highly likely to contain a Type I DNA restriction and modification (RM) system. The RM system usually destroys the invading DNA. Some of the anti-restriction proteins are DNA mimics and bind to the RM enzyme to prevent it binding to DNA. In this article, we characterize ArdB anti-restriction proteins and their close homologues, the KlcA proteins from a range of mobile genetic elements; including an ArdB encoded on a pathogenicity island from uropathogenic Escherichia coli and a KlcA from an IncP-1b plasmid, pBP136 isolated from Bordetella pertussis. We show that all the ArdB and KlcA act as anti-restriction proteins and inhibit the four main families of Type I RM systems in vivo, but fail to block the restriction endonuclease activity of the archetypal Type I RM enzyme, EcoKI, in vitro indicating that the action of ArdB is indirect and very different from that of the DNA mimics. We also present the structure determined by NMR spectroscopy of the pBP136 KlcA protein. The structure shows a novel protein fold and it is clearly not a DNA structural mimic.
doi:10.1093/nar/gkp1144
PMCID: PMC2836571  PMID: 20007596
14.  Extensive DNA mimicry by the ArdA anti-restriction protein and its role in the spread of antibiotic resistance 
Nucleic Acids Research  2009;37(15):4887-4897.
The ardA gene, found in many prokaryotes including important pathogenic species, allows associated mobile genetic elements to evade the ubiquitous Type I DNA restriction systems and thereby assist the spread of resistance genes in bacterial populations. As such, ardA contributes to a major healthcare problem. We have solved the structure of the ArdA protein from the conjugative transposon Tn916 and find that it has a novel extremely elongated curved cylindrical structure with defined helical grooves. The high density of aspartate and glutamate residues on the surface follow a helical pattern and the whole protein mimics a 42-base pair stretch of B-form DNA making ArdA by far the largest DNA mimic known. Each monomer of this dimeric structure comprises three alpha–beta domains, each with a different fold. These domains have the same fold as previously determined proteins possessing entirely different functions. This DNA mimicry explains how ArdA can bind and inhibit the Type I restriction enzymes and we demonstrate that 6 different ardA from pathogenic bacteria can function in Escherichia coli hosting a range of different Type I restriction systems.
doi:10.1093/nar/gkp478
PMCID: PMC2731889  PMID: 19506028
15.  Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping 
Nucleic Acids Research  2009;37(6):2053-2063.
Atomic force microscopy (AFM) allows the study of single protein–DNA interactions such as those observed with the Type I Restriction–Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization. Dimerization is particularly common if the DNA molecule contains two EcoKI recognition sites. DNA loops with dimers at their apex form if the DNA is sufficiently long, and also form in the presence of ATPγS, a non-hydrolysable analogue of the ATP required for translocation, indicating that the looping is on the reaction pathway of the enzyme. Visualization of specific DNA loops in the protein–DNA constructs was achieved by improved sample preparation and analysis techniques. The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping.
doi:10.1093/nar/gkp042
PMCID: PMC2665228  PMID: 19223329
16.  The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein 
Nucleic Acids Research  2008;37(3):762-770.
Type-I DNA restriction–modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M2S1 methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years.
doi:10.1093/nar/gkn988
PMCID: PMC2647291  PMID: 19074193
17.  Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes 
Nucleic Acids Research  2005;33(22):6953-6960.
DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 Å resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (∼100 ps) decay component and the large increase in the amplitude of the long (∼10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA–enzyme complexes that cannot be discerned from the present X-ray structures.
doi:10.1093/nar/gki995
PMCID: PMC1310896  PMID: 16340006
18.  DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping 
Nucleic Acids Research  2005;33(10):3235-3244.
The maintenance methyltransferase M.EcoKI recognizes the bipartite DNA sequence 5′-AACNNNNNNGTGC-3′, where N is any nucleotide. M.EcoKI preferentially methylates a sequence already containing a methylated adenine at or complementary to the underlined bases in the sequence. We find that the introduction of a single-stranded gap in the middle of the non-specific spacer, of up to 4 nt in length, does not reduce the binding affinity of M.EcoKI despite the removal of non-sequence-specific contacts between the protein and the DNA phosphate backbone. Surprisingly, binding affinity is enhanced in a manner predicted by simple polymer models of DNA flexibility. However, the activity of the enzyme declines to zero once the single-stranded region reaches 4 nt in length. This indicates that the recognition of methylation of the DNA is communicated between the two methylation targets not only through the protein structure but also through the DNA structure. Furthermore, methylation recognition requires base flipping in which the bases targeted for methylation are swung out of the DNA helix into the enzyme. By using 2-aminopurine fluorescence as the base flipping probe we find that, although flipping occurs for the intact duplex, no flipping is observed upon introduction of a gap. Our data and polymer model indicate that M.EcoKI bends the non-specific spacer and that the energy stored in a double-stranded bend is utilized to force or flip out the bases. This energy is not stored in gapped duplexes. In this way, M.EcoKI can determine the methylation status of two adenine bases separated by a considerable distance in double-stranded DNA and select the required enzymatic response.
doi:10.1093/nar/gki618
PMCID: PMC1143692  PMID: 15942026
19.  Alleviation of restriction by DNA condensation and non-specific DNA binding ligands 
Nucleic Acids Research  2004;32(19):5841-5850.
During conditions of cell stress, the type I restriction and modification enzymes of bacteria show reduced, but not zero, levels of restriction of unmethylated foreign DNA. In such conditions, chemically identical unmethylated recognition sequences also occur on the chromosome of the host but restriction alleviation prevents the enzymes from destroying the host DNA. How is this distinction between chemically identical DNA molecules achieved? For some, but not all, type I restriction enzymes, alleviation is partially due to proteolytic degradation of a subunit of the enzyme. We identify that the additional alleviation factor is attributable to the structural difference between foreign DNA entering the cell as a random coil and host DNA, which exists in a condensed nucleoid structure coated with many non-specific ligands. The type I restriction enzyme is able to destroy the ‘naked’ DNA using a complex reaction linked to DNA translocation, but this essential translocation process is inhibited by DNA condensation and the presence of non-specific ligands bound along the DNA.
doi:10.1093/nar/gkh918
PMCID: PMC528803  PMID: 15520467
20.  SURVEY AND SUMMARY: A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes 
Nucleic Acids Research  2003;31(7):1805-1812.
A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.
PMCID: PMC152790  PMID: 12654995
21.  Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes 
Research highlights
► Successful fusion of GFP to M.EcoKI DNA methyltransferase. ► GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. ► FRET confirms structural model of M.EcoKI bound to DNA.
We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Förster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.
doi:10.1016/j.bbrc.2010.06.069
PMCID: PMC2914225  PMID: 20599730
DNA restriction/modification; DNA methyltransferase; Forster resonance energy transfer; Time-resolved fluorescence anisotropy; Time-resolved fluorescence; Green fluorescent protein
22.  Mutations of the domain forming the dimeric interface of the ArdA protein affect dimerization and antimodification activity but not antirestriction activity 
The Febs Journal  2013;280(19):4903-4914.
ArdA antirestriction proteins are encoded by genes present in many conjugative plasmids and transposons within bacterial genomes. Antirestriction is the ability to prevent cleavage of foreign incoming DNA by restriction-modification (RM) systems. Antimodification, the ability to inhibit modification by the RM system, can also be observed with some antirestriction proteins. As these mobile genetic elements can transfer antibiotic resistance genes, the ArdA proteins assist their spread. The consequence of antirestriction is therefore the enhanced dissemination of mobile genetic elements. ArdA proteins cause antirestriction by mimicking the DNA structure bound by Type I RM enzymes. The crystal structure of ArdA showed it to be a dimeric protein with a highly elongated curved cylindrical shape [McMahon SA et al. (2009) Nucleic Acids Res37, 4887–4897]. Each monomer has three domains covered with negatively charged side chains and a very small interface with the other monomer. We investigated the role of the domain forming the dimer interface for ArdA activity via site-directed mutagenesis. The antirestriction activity of ArdA was maintained when up to seven mutations per monomer were made or the interface was disrupted such that the protein could only exist as a monomer. The antimodification activity of ArdA was lost upon mutation of this domain. The ability of the monomeric form of ArdA to function in antirestriction suggests, first, that it can bind independently to the restriction subunit or the modification subunits of the RM enzyme, and second, that the many ArdA homologues with long amino acid extensions, present in sequence databases, may be active in antirestriction.
Structured digital abstract
ArdA and ArdA bind by molecular sieving (1, 2)
ArdA and ArdA bind by cosedimentation in solution (1, 2)
doi:10.1111/febs.12467
PMCID: PMC3906837  PMID: 23910724
antirestriction; ArdA; horizontal gene transfer; restriction enzyme; Tn916
23.  Dissection of the DNA Mimicry of the Bacteriophage T7 Ocr Protein using Chemical Modification 
Journal of Molecular Biology  2009;391(3):565-576.
The homodimeric Ocr (overcome classical restriction) protein of bacteriophage T7 is a molecular mimic of double-stranded DNA and a highly effective competitive inhibitor of the bacterial type I restriction/modification system. The surface of Ocr is replete with acidic residues that mimic the phosphate backbone of DNA. In addition, Ocr also mimics the overall dimensions of a bent 24-bp DNA molecule. In this study, we attempted to delineate these two mechanisms of DNA mimicry by chemically modifying the negative charges on the Ocr surface. Our analysis reveals that removal of about 46% of the carboxylate groups per Ocr monomer results in an ∼ 50-fold reduction in binding affinity for a methyltransferase from a model type I restriction/modification system. The reduced affinity between Ocr with this degree of modification and the methyltransferase is comparable with the affinity of DNA for the methyltransferase. Additional modification to remove ∼ 86% of the carboxylate groups further reduces its binding affinity, although the modified Ocr still binds to the methyltransferase via a mechanism attributable to the shape mimicry of a bent DNA molecule. Our results show that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by up to ∼ 800-fold.
doi:10.1016/j.jmb.2009.06.020
PMCID: PMC2806950  PMID: 19523474
Ocr, overcome classical restriction; R/M, restriction/modification; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; HOBt, hydroxybenzotriazole; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; FT-ICR, Fourier transform ion cyclotron resonance; GdmCl, guanidinium hydrochloride; SAM, S-adenosyl-L-methionine; ITC, isothermal titration calorimetry; WT, wild type; DNA mimic; chemical modification; restriction/modification system
24.  How much of protein sequence space has been explored by life on Earth? 
We suggest that the vastness of protein sequence space is actually completely explorable during the populating of the Earth by life by considering upper and lower limits for the number of organisms, genome size, mutation rate and the number of functionally distinct classes of amino acids. We conclude that rather than life having explored only an infinitesimally small part of sequence space in the last 4 Gyr, it is instead quite plausible for all of functional protein sequence space to have been explored and that furthermore, at the molecular level, there is no role for contingency.
doi:10.1098/rsif.2008.0085
PMCID: PMC2459213  PMID: 18426772
protein sequence; evolution; contingency
25.  Differential stabilization of reaction intermediates: specificity checkpoints for M.EcoRI revealed by transient fluorescence and fluorescence lifetime studies 
Nucleic Acids Research  2008;36(9):2917-2925.
M.EcoRI, a bacterial sequence-specific S-adenosyl-l-methionine-dependent DNA methyltransferase, relies on a complex conformational mechanism to achieve its remarkable specificity, including DNA bending, base flipping and intercalation into the DNA. Using transient fluorescence and fluorescence lifetime studies with cognate and noncognate DNA, we have characterized several reaction intermediates involving the WT enzyme. Similar studies with a bending-impaired, enhanced-specificity M.EcoRI mutant show minimal differences with the cognate DNA, but significant differences with noncognate DNA. These results provide a plausible explanation of the way in which destabilization of reaction intermediates can lead to changes in substrate specificity.
doi:10.1093/nar/gkn131
PMCID: PMC2396439  PMID: 18385156

Results 1-25 (28)