Modification Subunits Research Articles

Novel Methyltransferase Recognition Motif Identified in Chania multitudinisentens RB-25T gen. nov., sp. nov.

DNA methylation, defined by the addition of a methyl group to adenine or cytosine bases in DNA catalyzed by DNA methyltransferases (MTases), is one of the most studied post-replicative DNA modification mechanism in bacteria (Roberts et al., 2003b). The three forms of nucleotide methylation identified to date are: N6-methyladenine(m6A), N4-methylcytosine (m4C), and 5-methylcytosine (m5C) (Gromova and Khoroshaev, 2003). Generally, MTases can be classified into two main groups: as part of a restriction modification (RM) system in which the MTase is associated with a cognate restriction endonuclease (REase) or as a solitary MTase, which as the name suggests, serves the role of an independent MTase (Murphy et al., 2013; Roberts et al., 2015). The RM systems, which are found to occur exclusively in unicellular organisms, can be further classified into four groups based on their subunit composition, recognition sequence specificity, substrate specificity, cofactor requirements, and DNA cleavage positions (Wilson and Murray, 1991; Casadesus and Low, 2006). In brief, Type I RM systems comprise three polypeptides which form a hetero-oligomeric protein complex, namely the R (restriction), M (modification), and S (specificity) subunits, that recognizes asymmetric, and bipartite recognition sequences (Murray, 2000). Type II systems, the most ubiquitous, and simplest systems, are composed of two functionally-independent R and M genes, which are responsible for restriction and methylation activity respectively. The recognition sequences of the Type II systems are most often symmetrical, but can also be asymmetric (Wilson and Murray, 1991). Type III system also consist of two subunits, named res, and mod. The mod subunit can function independently as a MTase, but only methylates one strand of the DNA. The res subunit must form a complex with the mod subunit to express its DNA restriction activity, because the recognition specificity is encoded in the mod subunit (Wilson and Murray, 1991). The recognition sequences of the known Type III systems are asymmetric and four to six bases in length. Furthermore, when known, the res enzyme requires the presence of two unmethylated recognition sites for efficient DNA cleavage (Rao et al., 2013). Lastly, the Type IV systems are restriction enzymes that recognize and cleave only methylated DNA (Vasu and Nagaraja, 2013). The sequence specificities of the Type IV systems are not well studied. MTases can be further sub-classified according to the order of their conserved amino acid motifs, which represent the DNA binding domain, the target recognition domain (TRD) and the catalytic domain (Malone et al., 1995; Jeltsch, 2002). RM systems are often considered as the most primitive defense mechanism of prokaryotes against the invasion of extraneous DNA elements (Wilson and Murray, 1991; Bickle, 2004). However, more recent studies have offered new insights into their additional biological roles, including genomic island stabilization, species identity maintenance, generating, and enhancing genomic diversity for host fitness and adaptability as well as the regulation of gene expression (Vasu and Nagaraja, 2013). The development of Single Molecule Real-Time (SMRT) sequencing technology which enables the simultaneous genome-wide detection of MTase activity during genome sequencing has permitted the rapid detection of novel MTase recognition motifs in various prokaryotes including Helicobacter pylori, Salmonella enterica, Escherichia coli, and Campylobacter coli (Krebes et al., 2014; Forde et al., 2015; Lee et al., 2015; Pirone-Davies et al., 2015; Zautner et al., 2015). These data are important not only for the identification of novel types of MTases, but they also lay the groundwork for the discovery of new biological roles for DNA methylation. Furthermore, these new data on previously characterized methylomes can further extend our understanding of these RM systems. Chania multitudinisentens RB-25T gen. nov., sp. nov. is a fully characterized newly proposed novel genus in the family of Enterobacteriaceae. Isolated in a soil sample collected from a former municipal landfill site, C. multitudinisentens RB-25T was initially misidentified as a member of the Serratia genus (Ee et al., 2014a). Further in-depth investigation later reclassified C. multitudinisentens RB-25T as a novel genus (Ee et al., 2016). To date, C. multitudinisentens RB-25T is only characterized for its quorum sensing properties (the production of C4-HSL, C6-HSL, and 3-oxo-C6-HSL) and its potential chitinolytic activity (Ee et al., 2014a; Lim et al., 2015b). In this study, we report a novel methyltransferase recognition motif in C. multitudinisentens RB-25T, which will serve as the foundation for future investigation of the role of methylation in the genus Chania.

Structural basis of asymmetric DNA methylation and ATP-triggered long-range diffusion by EcoP15I.

Type III R–M enzymes were identified >40 years ago and yet there is no structural information on these multisubunit enzymes. Here we report the structure of a Type III R–M system, consisting of the entire EcoP15I complex (Mod2Res1) bound to DNA. The structure suggests how ATP hydrolysis is coupled to long-range diffusion of a helicase on DNA, and how a dimeric methyltransferase functions to methylate only one of the two DNA strands. We show that the EcoP15I motor domains are specifically adapted to bind double-stranded DNA and to facilitate DNA sliding via a novel ‘Pin' domain. We also uncover unexpected ‘division of labour', where one Mod subunit recognizes DNA, while the other Mod subunit methylates the target adenine—a mechanism that may extend to adenine N6 RNA methylation in mammalian cells. Together the structure sheds new light on the mechanisms of both helicases and methyltransferases in DNA and RNA metabolism.

Type III restriction endonucleases are heterotrimeric: comprising one helicase–nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism.

Structural Insights into the Assembly and Shape of Type III Restriction–Modification (R–M) EcoP15I Complex by Small-Angle X-ray Scattering

EcoP15I is the prototype of the Type III restriction enzyme family, composed of two modification (Mod) subunits to which two (or one) restriction (Res) subunits are then added. The Mod subunits are responsible for DNA recognition and methylation, while the Res subunits are responsible for ATP hydrolysis and cleavage. Despite extensive biochemical and genetic studies, there is still no structural information on Type III restriction enzymes. We present here small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analysis of the EcoP15I holoenzyme and the Mod2 subcomplex. We show that the Mod2 subcomplex has a relatively compact shape with a radius of gyration (RG) of ∼37.4 Å and a maximal dimension of ∼110 Å. The holoenzyme adopts an elongated crescent shape with an RG of ∼65.3 Å and a maximal dimension of ∼218 Å. From reconstructed SAXS envelopes, we postulate that Mod2 is likely docked in the middle of the holoenzyme with a Res subunit at each end. We discuss the implications of our model for EcoP15I action, whereby the Res subunits may come together and form a “sliding clamp” around the DNA.

Mechanistic insights into type III restriction enzymes

Type III restriction-modification (R-M) enzymes need to interact with two separate unmethylated DNA sequences in indirectly repeated, head-to-head orientations for efficient cleavage to occur at a defined location next to only one of the two sites. However, cleavage of sites that are not in head-to-head orientation have been observed to occur under certain reaction conditions in vitro. ATP hydrolysis is required for the long-distance communication between the sites prior to cleavage. Type III R-M enzymes comprise two subunits, Res and Mod that form a homodimeric Mod2 and a heterotetrameric Res2Mod2 complex. The Mod subunit in M2 or R2M2 complex recognizes and methylates DNA while the Res subunit in R2M2 complex is responsible for ATP hydrolysis, DNA translocation and cleavage. A vast majority of biochemical studies on Type III R-M enzymes have been undertaken using two closely related enzymes, EcoP1I and EcoP15I. Divergent opinions about how the long-distance interaction between the recognition sites exist and at least three mechanistic models based on 1D- diffusion and/or 3D- DNA looping have been proposed.

DNA looping and translocation provide an optimal cleavage mechanism for the type III restriction enzymes

EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I-DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for restriction by type III restriction enzymes and highlight the similarities between this and other classes of restriction enzymes.

A Mutation in the Mod Subunit of EcoP15I Restriction Enzyme Converts the DNA Methyltransferase to a Site-specific Endonuclease

A closer inspection of the amino acid sequence of EcoP15I DNA methyltransferase revealed a region of similarity to the PDXn(D/E)XK catalytic site of type II restriction endonucleases, except for methionine in EcoP15I DNA methyltransferase instead of proline. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. EcoP15I-M357P DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically EcoP151-M357P.DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically, 5'-CAGCAG(N)(10)-3', as indicated by the arrows, in presence of magnesium ions.

Structural Domains in the Type III Restriction Endonuclease EcoP15I: Characterization by Limited Proteolysis, Mass Spectrometry and Insertional Mutagenesis

The Type III restriction endonuclease EcoP15I forms a hetero-oligomeric enzyme complex that consists of two modification (Mod) subunits and two restriction (Res) subunits. Structural data on Type III restriction enzymes in general are lacking because of their remarkable size of more than 400 kDa and the laborious and low-yield protein purification procedures. We took advantage of the EcoP15I-overexpressing vector pQEP15 and affinity chromatography to generate a quantity of EcoP15I high enough for comprehensive proteolytic digestion studies and analyses of the proteolytic fragments by mass spectrometry. We show here that in the presence of specific DNA the entire Mod subunit is protected from trypsin digestion, whereas in the absence of DNA stable protein domains of the Mod subunit were not detected. In contrast, the Res subunit is comprised of two trypsin-resistant domains of approximately 77−79 kDa and 27−29 kDa, respectively. The cofactor ATP and the presence of DNA, either specific or unspecific, are important stabilizers of the Res subunit. The large N-terminal domain of Res contains numerous functional motifs that are predicted to be involved in ATP-binding and hydrolysis and/or DNA translocation. The C-terminal small domain harbours the catalytic center. Based on our data, we conclude that both structural Res domains are connected by a flexible linker region that spans 23 amino acid residues. To confirm this conclusion, we have investigated several EcoP15I enzyme mutants obtained by insertion mutagenesis in and around the predicted linker region within the Res subunit. All mutants tolerated the genetic manipulation and did not display loss of function or alteration of the DNA cleavage position.

S-adenosyl-l-methionine is required for DNA cleavage by type III restriction enzymes

The requirement of S-adenosyl-l-methionine (AdoMet) in the cleavage reaction carried out by type III restriction-modification enzymes has been investigated. We show that DNA restriction by EcoPI restriction enzyme does not take place in the absence of exogenously added AdoMet. Interestingly, the closely related EcoP15I enzyme has endogenously bound AdoMet and therefore does not require the addition of the cofactor for DNA cleavage. By employing a variety of AdoMet analogs, which differ structurally from AdoMet, this study demonstrates that the carboxyl group and any substitution at the epsilon carbon of methionine is absolutely essential for DNA cleavage. Such analogs could bring about the necessary conformational change(s) in the enzyme, which make the enzyme proficient in DNA cleavage. Our studies, which include native polyacrylamide gel electrophoresis, molecular size exclusion chromatography, UV, fluorescence and circular dichroism spectroscopy, clearly demonstrate that the holoenzyme and apoenzyme forms of EcoP15I restriction enzyme have different conformations. Furthermore, the Res and Mod subunits of the EcoP15I restriction enzyme can be separated by gel filtration chromatography in the presence of 2 M NaCl. Reconstitution experiments, which involve mixing of the isolated subunits, result in an apoenzyme form, which is restriction proficient in the presence of AdoMet. However, mixing the Res subunit with Mod subunit deficient in AdoMet binding does not result in a functional restriction enzyme. These observations are consistent with the fact that AdoMet is required for DNA cleavage. In vivo complementation of the defective mod allele with a wild-type mod allele showed that an active restriction enzyme could be formed. Furthermore, we show that while the purified c2-134 mutant restriction enzyme is unable to cleave DNA, the c2-440 mutant enzyme is able to cleave DNA albeit poorly. Taken together, these results suggest that AdoMet binding causes conformational changes in the restriction enzyme and is necessary to bring about DNA cleavage.

LlaFI, a type III restriction and modification system in Lactococcus lactis.

We describe a type III restriction and modification (R/M) system, LlaFI, in Lactococcus lactis. LlaFI is encoded by a 12-kb native plasmid, pND801, harbored in L. lactis LL42-1. Sequencing revealed two adjacent open reading frames (ORFs). One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide. The two ORFs appear to be organized in an operon. A homology search revealed that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits. The complete amino acid sequence of the Mod subunit of LlaFI was aligned with the amino acid sequences of four previously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable. An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein. The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the LlaFI Res sequence. LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzymes. ATP and Mg2+ are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it. To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.