Cloning and expression of the BalI restriction-modification system.

BalI, a type II restriction-modification (R-M) system from the bacterium, Brevibacterium albidum, recognizes the DNA sequence 5'-TGGCCA-3'. We cloned the genes encoding the BalI restriction endonuclease and methyltransferase and expressed them in Escherichia coli. The two genes were aligned tail-to-tail and their termination codons overlapped. BalI restriction endonuclease and methyltransferase comprise 260 and 280 amino acids, respectively, and have molecular weights of 29 043 and 31 999 Da. The amino acid sequence of BalI methyltransferase is similar to that of other m6A MTases, although it has been categorized as a m5C methyltransferase. A high expression system for the BalI restriction endonuclease was constructed in E. coli for the production of large quantities of enzyme.     

Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.     

Oligomeric Structure Diversity within the GIY-YIG Nuclease Family

The GIY-YIG nuclease domain has been identified in homing endonucleases, DNA repair and recombination enzymes, and restriction endonucleases. The Type II restriction enzyme Eco29kI belongs to the GIY-YIG nuclease superfamily and, like most of other family members, including the homing endonuclease I-TevI, is a monomer. It recognizes the palindromic sequence 5′-CCGC/GG-3′ (“/” marks the cleavage position) and cuts it to generate 3′-staggered ends. The Eco29kI monomer, which contains a single active site, either has to nick sequentially individual DNA strands or has to form dimers or even higher-order oligomers upon DNA binding to make a double-strand break at its target site. Here, we provide experimental evidence that Eco29kI monomers dimerize on a single cognate DNA molecule forming the catalytically active complex. The mechanism described here for Eco29kI differs from that of Cfr42I isoschisomer, which also belongs to the GIY-YIG family but is functional as a tetramer. This novel mechanism may have implications for the function of homing endonucleases and other enzymes of the GIY-YIG family.     

Construction of an overproducing strain,purification, and biochemical characterization of the 6His-Eco29kI restriction endonuclease

We constructed a strain of Escherichia coli overproducing 6His-tagged Eco29kI by placing the coding sequence under control of a strong bacteriophage T5 promoter. The yield of 6His-Eco29kI restriction endonuclease expression could be increased to about 20% of the total cellular protein, but inclusion bodies formed consisting of insoluble 6His-Eco29kI protein. We developed a fast and effective protocol for purification of the homogeneous enzyme from both soluble and insoluble fractions and established their identity by catalytic activity assay. The isolated enzymes were tested for recognition specificity and optimal reaction conditions as a function of NaCl and KCl concentrations, temperature, and pH compared with the native Eco29kI restriction endonuclease. The 6His-tagged enzyme retained the specificity of the native protein but had an altered optimum of its catalytic reaction.     

BstNI同功酶限制—修饰系统基因的表达检测和定位分析

鉴定了Ｅ．ｃｏｌｉ ＨＢ１０１和ＪＭ１１０的部分遗传标记,作为受体菌分别用于ＢｓｔＮＩ同功酶限制－修饰系统中限制性内切酶（Ｒ）基因和甲基化酶（Ｍ）基因表达的检测。用外切酶Ⅲ单向删切含Ｒ－Ｍ基因的ＤＮＡ片段,获得２３个缺失突变亚克隆。通过检测各亚克隆表达的Ｒ酶和Ｍ酶活性,将Ｒ和Ｍ基因分别定位在距克隆位点ＰｓｔＩ和０．２→１．４ｋｂ和１．５→３．３ｋｂ范围内。分析表明：该系统属于Ⅱ类限制－修饰系统,两     

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.     

The Neisseria gonorrhoeae S.NgoVIII restriction/modification system: a type IIs system homologous to the Haemophilus parahaemolyticus HphI restriction/modification system.

Strains of Neisseria gonorrhoeae possess numerous restriction-modification (R-M) systems. One of these systems, which has been found in all strains tested, encodes the S. NgoVIII specificity (5'TCACC 3') R-M system. We cloned two adjacent methyltransferase genes (dcmH and damH), each encoding proteins whose actions protect DNA from digestion by R.HphI or R.Ngo BI (5'TCACC 3'). The damH gene product is a N 6-methyladenine methyltransferase that recognizes this sequence. We constructed a plasmid containing multiple copies of the S.NgoVIII sequence, grew it in the presence of damH and used the HPLC to demonstrate the presence of N 6-methyladenine in the DNA. A second plasmid, containing overlapping damH and Escherichia coli dam recognition sequences in combination with various restriction digests, was used to identify which adenine in the recognition sequence was modified by damH. The predicted dcmH gene product is homologous to 5-methylcytosine methyltransferases. The products of both the dcmH and damH genes, as well as an open reading frame downstream of the damH gene are highly similar to the Haemophilus parahaemolyticus hphIMC , hphIMA and hphIR gene products, encoding the Hph I Type IIs R-M system. The S.NgoVIII R-M genes are flanked by a 97 bp direct repeat that may be involved in the mobility of this R-M system.     

Purification of the novel endonuclease, Hpy188I, and cloning of its restriction-modification genes reveal evidence of its horizontal transfer to the Helicobacter pylori genome

We have isolated a novel restriction endonuclease, Hpy188I, from Helicobacter pylori strain J188. Hpy188I recognizes the unique sequence, TCNGA, and cleaves the DNA between nucleotides N and G in its recognition sequence to generate a one-base 3' overhang. Cloning and sequence analysis of the Hpy188I modification gene in strain J188 reveal that hpy188IM has a 1299-base pair (bp) open reading frame (ORF) encoding a 432-amino acid product. The predicted protein sequence of M.Hpy188I contains conserved motifs typical of aminomethyltransferases, and Western blotting indicates that it is an N-6 adenine methyltransferase. Downstream of hpy188IM is a 513-bp ORF encoding a 170-amino acid product, that has a 41-bp overlap with hpy188IM. The predicted protein sequence from this ORF matches the amino acid sequence obtained from purified Hpy188I, indicating that it encodes the endonuclease. The Hpy188I R-M genes are not present in either strain of H. pylori that has been completely sequenced but are found in two of 11 H. pylori strains tested. The significantly lower G + C content of the Hpy188I R-M genes implies that they have been introduced relatively recently during the evolution of the H. pylori genome.     

MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

MmeI is an unusual Type II restriction enzyme that is useful for generating long sequence tags. We have cloned the MmeI restriction-modification (R-M) system and found it to consist of a single protein having both endonuclease and DNA methyltransferase activities. The protein comprises an amino-terminal endonuclease domain, a central DNA methyltransferase domain and C-terminal DNA recognition domain. The endonuclease cuts the two DNA strands at one site simultaneously, with enzyme bound at two sites interacting to accomplish scission. Cleavage occurs more rapidly than methyl transfer on unmodified DNA. MmeI modifies only the adenine in the top strand, 5′-TCCRAC-3′. MmeI endonuclease activity is blocked by this top strand adenine methylation and is unaffected by methylation of the adenine in the complementary strand, 5′-GTYGGA-3′. There is no additional DNA modification associated with the MmeI R-M system, as is required for previously characterized Type IIG R-M systems. The MmeI R-M system thus uses modification on only one of the two DNA strands for host protection. The MmeI architecture represents a minimal approach to assembling a restriction-modification system wherein a single DNA recognition domain targets both the endonuclease and DNA methyltransferase activities.     

Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily

Background

The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally.

Results

Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L) and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme.

Conclusion

Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our prediction that R.Eco29kI belongs to the GIY-YIG superfamily of nucleases. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD-(D/E)XK or HNH superfamilies of nucleases, and is instead a member of the unrelated GIY-YIG superfamily.