Fused eco29kIR- and M genes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems

<p>Abstract</p> <p>Background</p> <p>The discovery of restriction endonucleases and modification DNA methyltransferases, key instruments of genetic engineering, opened a new era of molecular biology through development of the recombinant DNA technology. Today, the number of potential proteins assigned to type II restriction enzymes alone is beyond 6000, which probably reflects the high diversity of evolutionary pathways. Here we present experimental evidence that a new type IIC restriction and modification enzymes carrying both activities in a single polypeptide could result from fusion of the appropriate genes from preexisting bipartite restriction-modification systems.</p> <p>Results</p> <p>Fusion of <it>eco29kIR </it>and <it>M </it>ORFs gave a novel gene encoding for a fully functional hybrid polypeptide that carried both restriction endonuclease and DNA methyltransferase activities. It has been placed into a subclass of type II restriction and modification enzymes - type IIC. Its MTase activity, 80% that of the M.Eco29kI enzyme, remained almost unchanged, while its REase activity decreased by three times, concurrently with changed reaction optima, which presumably can be caused by increased steric hindrance in interaction with the substrate. <it>In vitro </it>the enzyme preferentially cuts DNA, with only a low level of DNA modification detected. <it>In vivo </it>new RMS can provide a 10²-fold less protection of host cells against phage invasion.</p> <p>Conclusions</p> <p>We propose a molecular mechanism of appearing of type IIC restriction-modification and M.SsoII-related enzymes, as well as other multifunctional proteins. As shown, gene fusion could play an important role in evolution of restriction-modification systems and be responsible for the enzyme subclass interconversion. Based on the proposed approach, hundreds of new type IIC enzymes can be generated using head-to-tail oriented type I, II, and III restriction and modification genes. These bifunctional polypeptides can serve a basis for enzymes with altered recognition specificities. Lastly, this study demonstrates that protein fusion may change biochemical properties of the involved enzymes, thus giving a starting point for their further evolutionary divergence.</p

Alteration of Sequence Specificity of the Type IIS Restriction Endonuclease BtsI

The Type IIS restriction endonuclease BtsI recognizes and digests at GCAGTG(2/0). It comprises two subunits: BtsIA and BtsIB. The BtsIB subunit contains the recognition domain, one catalytic domain for bottom strand nicking and part of the catalytic domain for the top strand nicking. BtsIA has the rest of the catalytic domain that is responsible for the DNA top strand nicking. BtsIA alone has no activity unless it mixes with BtsIB to reconstitute the BtsI activity. During characterization of the enzyme, we identified a BtsIB mutant R119A found to have a different digestion pattern from the wild type BtsI. After characterization, we found that BtsIB(R119A) is a novel restriction enzyme with a previously unreported recognition sequence CAGTG(2/0), which is named as BtsI-1. Compared with wild type BtsI, BtsI-1 showed different relative activities in NEB restriction enzyme reaction buffers NEB1, NEB2, NEB3 and NEB4 and less star activity. Similar to the wild type BtsIB subunit, the BtsI-1 B subunit alone can act as a bottom nicking enzyme recognizing CAGTG(-/0). This is the first successful case of a specificity change among this restriction endonuclease type

Identification of new homologs of PD-(D/E)XK nucleases by support vector machines trained on data derived from profile–profile alignments

PD-(D/E)XK nucleases, initially represented by only Type II restriction enzymes, now comprise a large and extremely diverse superfamily of proteins. They participate in many different nucleic acids transactions including DNA degradation, recombination, repair and RNA processing. Different PD-(D/E)XK families, although sharing a structurally conserved core, typically display little or no detectable sequence similarity except for the active site motifs. This makes the identification of new superfamily members using standard homology search techniques challenging. To tackle this problem, we developed a method for the detection of PD-(D/E)XK families based on the binary classification of profile–profile alignments using support vector machines (SVMs). Using a number of both superfamily-specific and general features, SVMs were trained to identify true positive alignments of PD-(D/E)XK representatives. With this method we identified several PFAM families of uncharacterized proteins as putative new members of the PD-(D/E)XK superfamily. In addition, we assigned several unclassified restriction enzymes to the PD-(D/E)XK type. Results show that the new method is able to make confident assignments even for alignments that have statistically insignificant scores. We also implemented the method as a freely accessible web server at http://www.ibt.lt/bioinformatics/software/pdexk/

Creation of a type IIS restriction endonuclease with a long recognition sequence

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases

Genome comparison and context analysis reveals putative mobile forms of restriction–modification systems and related rearrangements

The mobility of restriction–modification (RM) gene complexes and their association with genome rearrangements is a subject of active investigation. Here we conducted systematic genome comparisons and genome context analysis on fully sequenced prokaryotic genomes to detect RM-linked genome rearrangements. RM genes were frequently found to be linked to mobility-related genes such as integrase and transposase homologs. They were flanked by direct and inverted repeats at a significantly high frequency. Insertion by long target duplication was observed for I, II, III and IV restriction types. We found several RM genes flanked by long inverted repeats, some of which had apparently inserted into a genome with a short target duplication. In some cases, only a portion of an apparently complete RM system was flanked by inverted repeats. We also found a unit composed of RM genes and an integrase homolog that integrated into a tRNA gene. An allelic substitution of a Type III system with a linked Type I and IV system pair, and allelic diversity in the putative target recognition domain of Type IIG systems were observed. This study revealed the possible mobility of all types of RM systems, and the diversity in their mobility-related organization

TstI, a Type II restriction–modification protein with DNA recognition, cleavage and methylation functions in a single polypeptide

Type II restriction–modification systems cleave and methylate DNA at specific sequences. However, the Type IIB systems look more like Type I than conven-tional Type II schemes as they employ the same pro-tein for both restriction and modification and for DNA recognition. Several Type IIB proteins, including the archetype BcgI, are assemblies of two polypeptides: one with endonuclease and methyltransferase roles, another for DNA recognition. Conversely, some IIB proteins express all three functions from separate segments of a single polypeptide. This study anal-ysed one such single-chain protein, TstI. Compar-ison with BcgI showed that the one- and the two-polypeptide systems differ markedly. Unlike the het-erologous assembly of BcgI, TstI forms a homote-tramer. The tetramer bridges two recognition sites before eventually cutting the DNA in both strands on both sides of the sites, but at each site the first double-strand break is made long before the second. In contrast, BcgI cuts all eight target bonds at two sites in a single step. TstI also differs from BcgI in either methylating or cleaving unmodified sites at similar rates. The site may thus be modified before TstI can make the second double-strand break. TstI MTase acts best at hemi-methylated sites

S-adenosyl methionine prevents promiscuous DNA cleavage by the EcoP1I type III restriction enzyme

DNA cleavage by the type III restriction endonuclease EcoP1I was analysed on circular and catenane DNA in a variety of buffers with different salts. In the presence of the cofactor S-adenosyl methionine (AdoMet), and irrespective of buffer, only substrates with two EcoP1I sites in inverted repeat were susceptible to cleavage. Maximal activity was achieved at a Res2Mod2 to site ratio of approximately 1:1 yet resulted in cleavage at only one of the two sites. In contrast, the outcome of reactions in the absence of AdoMet was dependent upon the identity of the monovalent buffer components, in particular the identity of the cation. With Na+, cleavage was observed only on substrates with two sites in inverted repeat at elevated enzyme to site ratios (>15:1). However, with K+ every substrate tested was susceptible to cleavage above an enzyme to site ratio of approximately 3:1, including a DNA molecule with two directly repeated sites and even a DNA molecule with a single site. Above an enzyme to site ratio of 2:1, substrates with two sites in inverted repeat were cleaved at both cognate sites. The rates of cleavage suggested two separate events: a fast primary reaction for the first cleavage of a pair of inverted sites; and an order-of-magnitude slower secondary reaction for the second cleavage of the pair or for the first cleavage of all other site combinations. EcoP1I enzymes mutated in either the ATPase or nuclease motifs did not produce the secondary cleavage reactions. Thus, AdoMet appears to play a dual role in type III endonuclease reactions: Firstly, as an allosteric activator, promoting DNA association; and secondly, as a "specificity factor", ensuring that cleavage occurs only when two endonucleases bind two recognition sites in a designated orientation. However, given the right conditions, AdoMet is not strictly required for DNA cleavage by a type III enzyme