Determination of methylation site of DNA-methyltransferase NlaX by a hybrid method.

Using a new method based on a combination of bisulfite reaction, the repair enzyme uracil-DNA glycosylase, and synthetic oligodeoxyribonucleotides, the methylation site of DNA-methyltransferase NlaX (M.NlaX) from Neisseria lactamica was established to be the inner cytosine in the double-stranded pentanucleotide recognition sequence 5'-CCNGG-3' (where N = any nucleoside). 5-Methylcytosine (m5C) type modification by M-N1aX was confirmed by the use of oligonucleotide substrates that contain 5-fluoro-2'-deoxycytidine.

Characterization of the type IV restriction modification system BspLU11III from Bacillus sp. LU11.

We report the characterization and cloning of the genes for an unusual type IV restriction-modification system, BspLU11III, from Bacillus sp. LU11. The system consists of two methyltransferases and one endonuclease, which also possesses methyltransferase activity. The three genes of the restriction-modification system, bsplu11IIIMa, bsplu11IIIMb and bsplu11IIIR, are closely linked and tandemly arranged. The corresponding enzymes recognize the dsDNA sequence 5'-GGGAC-3'/5'-GTCCC-3', with M.BspLU11IIIa modifying the A (underlined) of one strand and M.BspLU11IIIb the inner C (underlined) of the other strand. R.BspLU11III has both endonuclease and adenine-specific methyltransferase activities and is able to protect the DNA against cleavage by itself. In contrast to all type IV restriction-modification systems described so far, which have only one adenine-specific methyltransferase, BspLU11III is the first type IV restriction-modification system that includes two methyltransferases, one of them being cytosine specific.

Determination of a non-methylated deoxycytidine residue in the recognition site of DNA-methyltransferases.

A method for determination of a non-methylated deoxycytidine (dC) residue in the recognition site of 5-cytosine DNA-methyltransferases is suggested. The method is based on treatment of methylated DNA by sodium bisulfite and successive reaction of the thus modified DNA with a repair enzyme, uracil-DNA glycosylase. This method was successfully applied to identify NlaX methyltransferase specificity.

Shape and DNA packaging activity of bacteriophage SPP1 procapsid: protein components and interactions during assembly.

The procapsid of the Bacillus subtilis bacteriophage SPP1 is formed by the major capsid protein gp13, the scaffolding protein gp11, the portal protein gp6, and the accessory protein gp7. The protein stoichiometry suggests a T=7 symmetry for the SPP1 procapsid. Overexpression of SPP1 procapsid proteins in Escherichia coli leads to formation of biologically active procapsids, procapsid-like, and aberrant structures. Co-production of gp11, gp13 and gp6 is essential for assembly of procapsids competent for DNA packaging in vitro. Presence of gp7 in the procapsid increases the yield of viable phages assembled during the reaction in vitro five- to tenfold. Formation of closed procapsid-like structures requires uniquely the presence of the major head protein and the scaffolding protein. The two proteins interact only when co-produced but not when mixed in vitro after separate synthesis. Gp11 controls the polymerization of gp13 into normal (T=7) and small sized (T=4?) procapsids. Predominant formation of T=7 procapsids requires presence of the portal protein. This implies that the portal protein has to be integrated at an initial stage of the capsid assembly process. Its presence, however, does not have a detectable effect on the rate of procapsid assembly during SPP1 infection. A stable interaction between gp6 and the two major procapsid proteins was only detected when the three proteins are co-produced. Efficient incorporation of a single portal protein in the procapsid appears to require a structural context created by gp11 and gp13 early during assembly, rather than strong interactions with any of those proteins. Gp7, which binds directly to gp6 both in vivo and in vitro, is not necessary for incorporation of the portal protein in the procapsid structure.

Structure of the 13-fold symmetric portal protein of bacteriophage SPP1.

We have determined the three-dimensional structure of bacteriophage SPP1 portal protein (gp6) using electron microscopy at liquid-helium temperatures and angular reconstitution. The 13-fold symmetric gp6 oligomer is a turbine-shaped structure with three distinct regions: a conical stem with a central channel; the turbine wings region; and a fringe of small 'tentacles' at the end of the channel exposed to the viral head interior. The tentacle region appears flexible and may be associated with a particular function - sensing when the correct amount of DNA has been packaged. The three-dimensional structure of the gp6 SizA mutant, which packages a smaller chromosome, reveals significant differences in that region.

M.(phi)BssHII, a novel cytosine-C5-DNA-methyltransferase with target-recognizing domains at separated locations of the enzyme.

In all cytosine-C5-DNA-methyltransferases (MTases) from prokaryotes and eukaryotes, remarkably conserved amino acid sequence elements responsible for general enzymatic functions are arranged in the same canonical order. In addition, one variable region, which includes the target-recognizing domain(s) (TRDs) characteristic for each enzyme, has been localized in one region between the same blocks of these conserved elements. This conservation in the order of conserved and variable sequences suggests stringent structural constraints in the primary structure to obtain the correct folding of the enzymes. Here we report the characterization of a new type of a multispecific MTase, M.(phiphi)BssHII, which is expressed as two isoforms. Isoform I is an entirely novel type of MTase which has, in addition to the TRDs at the conventional location, one TRD located at a non-canonical position at its N-terminus. Isoform II is represented by the same MTase, but without the N-terminal TRD. The N-terminal TRD provides HaeII methylation specificity to isoform I. The TRD is fully functional when engineered into either the conventional variable region of M.(phiphi)BssHII or the related monospecific M.phi3TII MTase. The implications of this structural plasticity with respect to the evolution of MTases are discussed.

A gene essential for de novo methylation and development in Ascobolus reveals a novel type of eukaryotic DNA methyltransferase structure.

Molecular mechanisms determining methylation patterns in eukaryotic genomes still remain unresolved. We have characterized, in Ascobolus, a gene for de novo methylation. This novel eukaryotic gene, masc1, encodes a protein that has all motifs of the catalytic domain of eukaryotic C5-DNA-methyltransferases but is unique in that it lacks a regulatory N-terminal domain. The disruption of masc1 has no effect on viability or methylation maintenance but prevents the de novo methylation of DNA repeats, which takes place after fertilization, through the methylation induced premeiotically (MIP) process. Crosses between parents harboring the masc1 disruption are arrested at an early stage of sexual reproduction, indicating that the activity of Masc1, the product of the gene, is crucial in this developmental process.

Head morphogenesis genes of the Bacillus subtilis bacteriophage SPP1.

We have identified and characterized the phage cistrons required for assembly of SPP1 heads. A DNA fragment containing most of the head morphogenesis genes was cloned and sequenced. The 3'-end of a previously identified gene (gene 6) and eight complete open reading frames (7 to 15) were predicted. We have assigned genes 7, 8, 9, 11, 12, 13, 14 and 15 to these orfs by correlating genetic and immunological data with DNA and protein sequence information. G7P was identified as a minor structural component of proheads and heads, G11P as the scaffold protein, G12P and G15P as head minor proteins and G13P as the coat protein. Characterization of intermediates in head assembly, which accumulate during infection with mutants deficient in DNA packaging or in morphogenetic genes, allowed the definition of the head assembly pathway. No proteolytic processing of any of the head components was detected. Removal of G11P by mutation leads to the accumulation of prohead-related structures and aberrant particles which are similar to the assemblies formed by purified G13P in the absence of other phage-encoded proteins. The native molecular masses of G11P and G13P are about 350 kDa and larger than 5000 kDa, respectively (predicted molecular masses 23.4 kDa and 35.3 kDa, respectively). G13P, upon denaturation and renaturation, assembles from protomers into some prohead-related structures. The organization of the DNA packaging and head genes of SPP1 resembles the organization of genes in the analogous regions of phage lambda and P22.

Masc2, a C5-DNA-methyltransferase from Ascobolus immersus with similarity to methyltransferases of higher organisms.

The filamentous fungus Ascobolus immersus represents an eukaryotic model organism to study genetic phenomena linked to DNA methylation. Following our previous characterization of a gene, masc1 from A. immersus, encoding the 'de novo' C5-DNA-methyltransferase (MTase), we report here the identification of a second MTase gene, masc2. The deduced peptide sequence of Masc2 is similar to previously identified eukaryotic MTases and distinct from Masc1 by having a large N-terminal domain in addition to the ubiquitous C-terminal catalytic domain. Following cloning of the gene, Masc2 was overexpressed and purified. Masc2 shows MTase activity with double stranded DNAs. Structural and biochemical properties of Masc2 suggest that it may function as a 'maintenance' MTase. With this finding, A. immersus represents so far the only eukaryotic organism in which two possibly synergistically operating MTases have been identified.

The complete nucleotide sequence and functional organization of Bacillus subtilis bacteriophage SPP1.

The complete nucleotide sequence of the B. subtilis bacteriophage SPP1 is described. The genome is 44,007 bp in size and has a base composition of 43.7% dG + dC. Only 32.2 kb are essential for phage amplification under laboratory conditions. Transcription using only the 'heavy strand' is asymmetric. Eighty-one orfs organized in five early and four late operons were identified. Experiments have shown that 25 orfs are essential. Of the remaining orfs, functions could be predicted for the products of five of the orfs on the basis of comparison of the deduced amino acid sequence to known proteins. Intergenic regions include most of the 5 PE and the 4 PL promoters. Transcripts are polycistronic. Transcription from the PE promoters is mediated by host RP, whereas recognition of the PL promoters requires an additional unidentified phage-encoded product. Translation of mRNA transcribed from most of the orfs seems to be initiated independently, each from its own ribosomal binding and initiation site, although a few cases of coupled translation have been reported. The organization of SPP1 genes involved in the replication, DNA packaging and phage assembly proteins resembles the organization of genes of equivalent regions of different E. coli double-stranded DNA phages. Absence of aa sequence similarity between analogous proteins of different phages suggested that the conserved gene organization is representative of a primordial bacteriophage.

Sequential headful packaging and fate of the cleaved DNA ends in bacteriophage SPP1.

The virulent Bacillus subtilis bacteriophage SPP1 packages its DNA from a precursor concatemer by a headful mechanism. Following disruption of mature virions with chelating agents the chromosome end produced by the headful cut remains stably bound to the phage tail. Cleavage of this tail-chromosome complex with restriction endonucleases that recognize single asymmetric positions within the SPP1 genome yields several distinct classes of DNA molecules whose size reflects the packaging cycle they were generated from. A continuous decrease in the number of molecules within each class derived from successive encapsidation rounds indicates that there are several packaging series which end after each headful packaging cycle. The frequency of molecules in each packaging class follows the distribution expected for a sequential mechanism initiated unidirectionally at a defined position in the genome (pac). The heterogeneity of the DNA fragment sizes within each class reveals an imprecision in headful cleavage of approximately 2.5 kb (5.6% of the genome size). The number of encapsidation events in a packaging series (processivity) was observed to increase with time during the infection process. DNA ejection through the tail can be induced in vitro by a variety of mild denaturing conditions. The first DNA extremity to exit the virion is invariably the same that was observed to be bound to the tail, implying that the viral chromosome is ejected with a specific polarity to penetrate the host. In mature virions a short segment of this chromosome end (55 to 67 bp equivalent to 187 to 288 A) is fixed to the tail area proximal to the head (connector). Upon ejection this extremity is the first to move along the tail tube to exit from the virion through the region where the tail spike was attached.

M.BssHII, a multispecific cytosine-C5-DNA-methyltransferase with unusual target recognizing properties.

A new multispecific cytosine-C5-DNA-methyltransferase (C5-MTase), M.BssHII, was identified in Bacillus stearothermophilus H3. The M.BssHII gene was cloned and sequenced. The amino acid sequence deduced shows the characteristic building plan of a C5-MTase. By sequencing bisulfite-treated DNA methylated by M.BssHII and by restriction enzyme analysis, we defined the following methylation targets of M.BssHII: ACGCGT/CCGCGG (MluI/SacII), PuGCGCPy (HaeII), PuCCGGPy (Cfr10I) and GCGCGC (BssHII). The relative location of the specificity determinants in the C5-MTase was derived from the analysis of M.BssHII derivatives carrying deletions within the variable region "V" and chimeric C5-Mtases constructed between M.BssHII and the related monospecific enzyme M.phi3TII. Four of the M.BssHII specificities (MluI, SacII, Cfr10I and BssHII) could be associated with amino acid segments within the variable region "V". The determinant for HaeII activity had to be assigned to sequences defining the enzyme core, the first example of a C5-MTase in which a sequence-specific methylation potential is mediated by structures outside of the variable region. Another intriguing result came from the analysis of one particular chimera made between M.BssHII and M.phi3TII. This construct showed a relaxation of the methylation capacity, both with respect to the target recognized and the targeting of methylation within this sequence.

Exact size and organization of DNA target-recognizing domains of multispecific DNA-(cytosine-C5)-methyltransferases.

A large portion of the sequences of type II DNA-(cytosine-C5)-methyltransferases (C5-MTases) represent highly conserved blocks of amino acids. General steps in the methylation reaction performed by C5-MTases have been found to be mediated by some of these domains. C5-MTases carry, in addition at the same relative location, a region variable in size and amino acid composition, part of which is associated with the capacity of each C5-MTase to recognize its characteristic target. Individual target-recognizing domains (TRDs) for the targets CCGG (M), CC(A/T)GG (E), GGCC (H), GCNGC (F) and G(G/A/T)GC(C/A/T)C (B) could be identified in the C-terminal part of the variable region of multispecific C5-MTases. With experiments reported here, we have established the organization of the variable regions of the multispecific MTases M.SPRI, M.phi3TI, M.H2I and M.rho 11SI at the resolution of individual amino acids. These regions comprise 204, 175, 268 and 268 amino acids, respectively. All variable regions are bipartite. They contain at their N-terminal side a very similar sequence of 71 amino acids. The integrity of this sequence must be assured to provide enzyme activity. Bracketed by 6-10 'linker' amino acids, they have, depending on the enzyme studied, towards their C-terminal end ensembles of individual TRDs of 38 (M), 39 (E), 40 (H), 44 (F) and 54 (B) amino acids. TRDs of different enzymes with equal specificity have the same size. TRDs do not overlap but are either separated by linker amino acids or abut each other.

Identification of a subdomain within DNA-(cytosine-C5)-methyltransferases responsible for the recognition of the 5' part of their DNA target.

In previous work on DNA-(cytosine-C5)-methyltransferases (C5-MTases), domains had been identified which are responsible for the sequence specificity of the different enzymes (target-recognizing domains, TRDs). Here we have analyzed the DNA methylation patterns of two C5-MTases containing reciprocal chimeric TRDs, consisting of the N- and C-terminal parts derived from two different parental TRDs specifying the recognition of 5'-CC(A/T)GG-3' and 5'-GCNGC-3'. Sequences recognized by these engineered MTases were non-symmetrical and degenerate, but contained at their 5' part a consensus sequence which was very similar to the 5' part of the target recognized by the parental TRD which contributed the N-terminal moiety of the chimeric TRD. The results are discussed in connection with the present understanding of the mechanism of DNA target recognition by C5-MTases. They demonstrate the possibility of designing C5-MTases with novel DNA methylation specificities.

M.BssHII: a new multispecific C5-DNA-methyltransferase.

M.BssHII is a new multispecific C5-DNA-methyltransferase recognizing five different targets. As the enzyme has been isolated from a thermophilic Bacillus, the protein should show enhanced intrinsic thermostability and therefore be a promising candidate for crystallizing a multispecific MTase.

Altered sequence recognition specificity of a C5-DNA methyltransferase carrying a chimeric 'target recognizing domain'.

A MTase with a chimeric TRD with the N-terminal half derived from a TRD recognizing GCNGC, the C-terminal half from one with CCWGG recognition, was constructed. Its target specificity is reported.

BsuCI, a type-I restriction-modification system in Bacillus subtilis.

A type-I R-M system was identified in B. subtilis. The genes comprising the system have striking similarity to type-I R-M systems observed in Enterobacteriaceae.

M.phi 3TII: a new monospecific DNA (cytosine-C5) methyltransferase with pronounced amino acid sequence similarity to a family of adenine-N6-DNA-methyltransferases.

The temperate B.subtilis phages phi 3T and rho 11s code, in addition to the multispecific DNA (cytosine-C5) methyltransferases (C5-MTases) M. phi 3TI and M. rho 11sI, which were previously characterized, for the identical monospecific C5-MTases M. phi 3TII and M. rho 11sII. These enzymes modify the C of TCGA sites, a novel target specificity among C5-MTases. The primary sequence of M. phi 3TII (326 amino acids) shows all conserved motifs typical of the building plan of C5-MTases. The degree of relatedness between M. phi 3TII and all other mono- or multispecific C5-MTases ranges from 30-40% amino acid identity. Particularly M. phi 3TII does not show pronounced similarity to M. phi 3TI indicating that both MTase genes were not generated from one another but were acquired independently by the phage. The amino terminal part of the M. phi 3TII (preceding the variable region 'V'), which predominantly constitutes the catalytic domain of the enzyme, exhibits pronounced sequence similarity to the amino termini of a family of A-N6-MTases, which--like M.TaqI--recognize the general sequence TNNA. This suggests that recently described similarities in the general three dimensional organization of C5- and A-N6-MTases imply divergent evolution of these enzymes originating from a common molecular ancestor.

M.phi 3TII: a new monospecific DNA (cytosine-C5) methyltransferase with pronounced amino acid sequence similarity to a family of adenine-N6-DNA-methyltransferases.

The temperate B.subtilis phages phi 3T and rho 11s code, in addition to the multispecific DNA (cytosine-C5) methyltransferases (C5-MTases) M.phi 3TI and M.rho 11sI, which were previously characterized, for the identical monospecific C5-MTases M.phi 3TII and M.rho 11sII. These enzymes modify the C to TCGA sites, a novel target specificity among C5-MTases. The primary sequence of M.phi 3TII (326 amino acids) shows all conserved motifs typical of the building plan of C5-MTases. The degree of relatedness between M.phi 3TII and all other mono- or multispecific C5-MTases ranges from 30-40% amino acid identity. Particularly M.phi 3TII does not show pronounced similarity to M.phi 3TI indicating that both MTase genes were not generated from one another but were acquired independently by the phage. The amino terminal part of the M.phi 3TII (preceding the variable region 'V'), which predominantly constitutes the catalytic domain of the enzyme, exhibits pronounced sequence similarity to the amino termini of a family of A-N6-MTases, which--like M.Taql--recognize the general sequence TNNA. This suggests that recently described similarities in the general three dimensional organization of C5- and A-N6-MTases imply divergent evolution of these enzymes originating from a common molecular ancestor.

Sequence analysis of the left end of the Bacillus subtilis bacteriophage SPP1 genome.

The left end of the genome of Bacillus subtilis bacteriophage SPP1 is represented by EcoRI DNA fragments 12 and 1 (EcoRI-12 and EcoRI-1). A number of different deletions were identified in EcoRI-1. A detailed physical and genetic map of EcoRI-1 from wild-type (wt) phage and SPP1 deletion mutants was constructed. Genes encoding essential products involved in late and early stages of phage DNA metabolism were mapped at the left and right ends of the 8.5-kb EcoRI-1, respectively. Deletions fell within the internal 5157-bp DNA segment of EcoRI-1. The nucleotide (nt) sequence of this region and of the endpoints of two deletions, delta X and delta L, were determined. The nt sequence of the junctions in SPP1 delta X and SPP1 delta L showed that, in these deletions, a segment of DNA between short directly repeated sequences of 10 and 13 bp, located 3427 and 4562 bp apart in the wt sequence, had been eliminated. In both cases, the copy of the repeated sequence was retained in the deletion mutant, consistent with the hypothesis that the deletions originated by homologous intramolecular recombination. The corresponding region in wt phage had fifteen presumptive open reading frames (orfs) and the previously identified SPP1 early promoters (PE1). The poor growth phenotype associated with the SPP1 deletion mutants was attributed to premature transcriptional read through from promoter(s) of the early region into late operon brought into close vicinity of the late genes due to the deletion event.