Cloning and characterization of M.LmoA118I, a novel DNA:m4C methyltransferase from the Listeria monocytogenes phage A118, a close homolog of M.NgoMXV.

A homolog of M.NgoMXV DNA:m4C methyltransferase has been identified among the open reading frames deduced from the genomic sequence of Listeria monocytogenes phage A118 [Loessner et al., 2000]. The gene coding for this putative protein has been cloned in Escherichia coli and its enzymatic activity in vivo in this host have been analyzed. Remarkably, despite M.NgoMXV and M.LmoA118I exhibit high sequence similarity (58% identical and 19% conservatively substituted residues), their target preferences differ: both proteins exhibit "relaxed" sequence specificity, but while M.LmoA118I more efficiently methylates GGCC sites, it seems to target only a subset of CCWGG sites methylated by M.NgoMXV.     

Structural characterization of two tandemly arranged DNA methyltransferase genes from Neisseria gonorrhoeae MS11: N4-cytosine specific M.NgoMXV and nonfunctional 5-cytosine-type M.NgoMorf2P

Two adjacent genes encoding DNA methyltransferases (MTases) of Neisseria gonorrhoeae MS11, an active N4-cytosine specific M. NgoMXV and an inactive 5-cytosine type M. NgoMorf2P, were cloned into Escherichia coli and sequenced. We analyzed the deduced amino acid sequence of both gene products and localized conserved regions characteristic for DNA MTases. Structure prediction, threading-derived alignments, and comparison with the common fold for DNA MTases allowed for construction of super-secondary and tertiary models for M.NgoMorf2P and M.NgoMXV, respectively. These models helped in identification of amino acids and structural elements essential for function of both enzymes. The implications of this putative structural model on the catalytic mechanism of M.NgoMXV and its possible relation to the common ancestor of modern DNA amino-MTases are also discussed.     

Analysis of the posttranslational modifications of the influenza virus M2 protein.

The sites of posttranslational modifications of the influenza A virus M2 protein were examined, and the effect of these modifications on the M2 protein ion channel activity was analyzed. Cysteine residues 17 and 19 in the M2 protein ectodomain form disulfide bonds. The cytoplasmic tail is posttranslationally modified by palmitoylation, and mutagenic studies support the view that cysteine residue 50 is the site for fatty acylation. In addition, the cytoplasmic tail of the M2 protein was found to be posttranslationally modified by the addition of phosphate to specific serine residues. Site-directed mutagenesis of serine residues in the M2 protein cytoplasmic tail, combined with phosphoamino acid analysis, indicated that serine residue 64 is the predominant site for phosphorylation but that serine residues 82, 89, and 93 were also phosphorylated but to much lesser extents. Disulfide-bond formation, palmitoylation, and phosphorylation occurred on M2 protein expressed in mammalian cells infected with influenza virus, in mammalian cells in which the M2 protein was expressed from DNA expression vectors, and when the M2 protein was expressed in oocytes of Xenopus laevis. The membrane currents of oocytes of Xenopus laevis expressing wild-type and site-specifically altered forms of the M2 protein, to ablate posttranslational modifications, indicated that none of the posttranslational modifications significantly affected the ion channel activity of the M2 protein in oocytes. Therefore, these data do not indicate a functional role for posttranslational modifications of the M2 protein in its ion channel activity.     

Control of simian virus 40 DNA replication by the HeLa cell nuclear kinase casein kinase I.

The initiation of simian virus 40 (SV40) DNA replication is regulated by the phosphorylation state of the viral initiator protein, large T antigen. We describe the purification from HeLa cell nuclei of a 35-kDa serine/threonine protein kinase that phosphorylates T antigen at sites that are phosphorylated in vivo and thereby inhibits its ability to initiate SV40 DNA replication. The inhibition of both origin unwinding and DNA replication by the kinase is reversed by protein phosphatase 2A. As determined by molecular weight, substrate specificity, autophosphorylation, immunoreactivity, and limited sequence analysis, this kinase appears to be identical to casein kinase I, a ubiquitous serine/threonine protein kinase that is closely related to a yeast kinase involved in DNA metabolism. The HeLa cell phosphorylation cycle that controls the initiation of SV40 DNA replication may also play a role in cellular DNA metabolism.     

Studies of Schizosaccharomyces pombe DNA polymerase alpha at different stages of the cell cycle.

The status of Schizosaccharomyces pombe (fission yeast) DNA polymerase alpha was investigated at different stages of the cell cycle. S.pombe DNA polymerase alpha is a phosphoprotein, with serine being the exclusive phosphoamino acid. By in vivo pulse labeling experiments DNA polymerase alpha was found to be phosphorylated to a 3-fold higher level in late S phase cells compared with cells in the G2 and M phases, but the steady-state level of phosphorylation did not vary significantly during the cell cycle. Tryptic phosphopeptide mapping demonstrated that the phosphorylation sites of DNA polymerase alpha from late S phase cells were not the same as that from G2/M phase cells. DNA polymerase alpha partially purified from G1/S cells had a different mobility in native gels from that from G2/M phase cells. The partially purified polymerase alpha from G1/S phase cells had a higher affinity for single-stranded DNA than that from G2/M phase cells. Despite the apparent differences in cell cycle-dependent phosphorylation, mobility in native gels and affinity for DNA, the in vitro enzymatic activity of the partially purified DNA polymerase alpha did not appear to vary during the cell cycle. The possible biological significance of these cell cycle-dependent characteristics of DNA polymerase alpha is discussed.     

The cysteine conserved among DNA cytosine methylases is required for methyl transfer, but not for specific DNA binding.

All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position. We show here that substitutions of this cysteine in the E. coli methylase M. EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA. Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA. These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor. Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate. However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding. We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA substrate for M. EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme. As expected, this modified substrate does not form irreversible complexes with the mutants.     

Enzymatic hydrolysis of p-nitroacetanilide: mechanistic studies of the aryl acylamidase from Pseudomonas fluorescens.

Aryl acylamidase (EC 3.1.5.13; AAA) catalyzes the hydrolysis of p-nitroacetanilide (PNAA) via the standard three-step mechanism of serine hydrolases: binding of substrate (K(s)), acylation of active-site serine (k(acyl)), and hydrolytic deacylation (k(deacyl)). Key mechanistic findings that emerged from this study include that (1) AAA requires a deprotonated base with a pK(a) of 8.3 for expression of full activity toward PNAA. Limiting values of kinetic parameters at high pH are k(c) = 7 s(-1), K(m) = 20 microM, and k(c)/K(m) = 340 000 M(-1) s(-1). (2) At pH 10, where all the isotope effects were conducted, k(c) is equally rate-limited by k(acyl) and k(deacyl). (3) The following isotope effects were determined: (D)()2(O)(k(c)/K(m)) = 1.7 +/- 0.2, (D)()2(O)k(c) = 3.5 +/- 0.3, and (beta)(D)(k(c)/K(m)) = 0.83 +/- 0.04, (beta)(D)k(c) = 0.96 +/- 0.01. These values, together with proton inventories for k(c)/K(m) and k(c), suggest the following mechanism: (i) The initial binding of substrate to enzyme to form the Michaelis complex is accompanied by solvation changes that generate solvent deuterium isotope effects originating from hydrogen ion fractionation at multiple sites on the enzyme surface. (ii) From within the Michaelis complex, the active site serine attacks the carbonyl carbon of PNAA with general-base catalysis to form a substantially tetrahedral transition state enroute to the acyl-enzyme. (iii) Finally, deacylation occurs through a process involving a rate-limiting solvent isotope effect, generating conformational change of the acyl-enzyme that positions the carbonyl bond in a polarizing environment that is optimal for attack by water.     

A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacterium.

PCR reactions were carried out on the genomic DNA of M. xanthus, a soil bacterium capable of differentiation to form fruiting bodies, using oligonucleotides representing highly conserved regions of eukaryotic protein serine/threonine kinases. A gene (pkn1) thus cloned contains an ORF of 693 amino acid residues whose amino-terminal domain shows significant sequence similarity with the catalytic domain of eukaryotic protein serine/threonine kinases. The pkn1 gene was overexpressed in E. coli, and the gene product has been found to be autophosphorylated at both serine and threonine residues. The expression of pkn1 is developmentally regulated to start immediately before spore formation. When pkn1 is deleted, differentiation starts prematurely, resulting in poor spore production. These results indicate that the protein serine/threonine kinase plays an important role in the onset of proper differentiation.     

Effect of Serine and Ethanolamine Administration on Phospholipid-Related Compounds and Neurotransmitter Amino Acids in the Rabbit Hippocampus

Abstract: The report concerns mechanisms for the increase of extracellular levels of ethanolamine and phosphoethanolamine in CNS regions, such as the hippocampus, in transient brain ischemia, hypoglycemia, seizures, etc. l -Serine (2.5–10 m M ), d -serine (10 m M ), or ethanolamine (10 m M ) was administered for 20 min via a microdialysis tubing to the hippocampus of unanesthetized rabbits. The concentrations of primary amines were determined in the dialysates. When levels were elevated 10–100 times in the extracellular fluid, l -serine caused a dose-dependent increase of the concentration of extracellular ethanolamine. Ethanolamine caused a corresponding, although somewhat smaller, increase in serine levels. Furthermore, l -serine also induced an increased concentration of phosphoethanolamine that was delayed in time relative to the peak of ethanolamine. d -Serine was as effective as l -serine in raising ethanolamine levels but had no effect on phosphoethanolamine. Ethanolamine, but not l -serine, also increased extracellular glutamate/aspartate levels in an MK-801-dependent fashion. A similar effect, but delayed in time, was observed with d -serine. These effects were inhibited by MK-801. The concentrations of other amino acids were not significantly affected. The characteristics of the effects are suggestive of base exchange reactions between serine and ethanolamine and between ethanolamine and serine glycerophospholipids, respectively, in neuronal plasma membranes.     

Identification of amino acids important for target recognition by the DNA:m5C methyltransferase M.NgoPII by alanine-scanning mutagenesis of residues at the protein-DNA interface

DNA:m(5)C MTases comprise a catalytic domain with conserved residues of the active site and a strongly diverged TRD with variable residues involved in DNA recognition and binding. To date, crystal structures of 2 DNA:m(5)C MTases complexed with the substrate DNA have been obtained; however, for none of these enzymes has the importance of the whole set of DNA-binding residues been comprehensively studied. We built a comparative model of M.NgoPII, a close homologue and isomethylomer of M.HaeIII, and systematically analyzed the effect of alanine substitutions for the complete set of amino acid residues from its TRD predicted to be important for DNA binding and target recognition. Our data demonstrate that only 1 Arg residue is indispensable for the MTase activity in vivo and in vitro, and that mutations of only a few other residues cause significant reduction of the activity in vitro, with little effect on the activity in vivo. The identification of dispensable protein-DNA contacts in the wild-type MTase will serve as a platform for exhaustive combinatorial mutagenesis aimed at the design of new contacts, and thus construction of enzyme variants that retain the activity but exhibit potentially new substrate preferences.     

Unusual properties of the DNA from Xanthomonas phage XP-12 in which 5-methylcytosine completely replaces cytosine.

Xanthomonas phage XP-12 contains 5-methylcytosine completely replacing cytosine. This substitution confers several unusual properties upon XP-12 DNA. The buoyant density of XP-12 DNA in CsCl gradients is 1.710 g/cm-3, 0.16 g/cm-3 lower than that expected for a normal DNA with the same percentage of adenine plus thymine. The melting temperature for XP-12 DNA in 0.012 M Na+ is the highest reported for any naturally occurring DNA, 83.2 degrees C, 6.1 degrees C higher than that of normal DNAs with the same percentage of adenine plus thymine. Unlike the minor amounts of 5-methylcytosine found in most plant and animal DNAs, the 5-methylcytosine residues of XP-12 derive their methyl group from the 3-carbon of serine instead of from the thiomethyl carbon of methionine. .     

DNA damage-dependent and -independent phosphorylation of the hRad9 checkpoint protein

Cell cycle checkpoints are regulatory mechanisms that maintain genomic integrity by preventing cell cycle progression when genetic anomalies are present. The hRad9 protein is the human homologue of Schizosaccharomyces pombe Rad9, a checkpoint protein required for preventing the onset of mitosis if DNA damage is present or if DNA replication is incomplete. Genetic and biochemical analyses indicate that hRad9 is a component of the checkpoint response in humans and has possible roles in regulating the cell cycle, apoptosis, and DNA repair. Previous studies indicate that hRad9 is modified by phosphorylation, both in the absence of exogenous stress and in response to various genotoxins. In this study, we report the mapping of several sites of constitutive phosphorylation of hRad9 to (S/T)PX(R/P) sequences near the C terminus of the protein. We also demonstrate that a serine to alanine mutation at residue 272 abrogates an ionizing radiation (IR)-induced phosphorylation of hRad9 and further show that phosphorylation at (S/T)P sites is not a prerequisite for IR-induced phosphorylation of serine 272. Finally, we report that hRad9 undergoes cell cycle-regulated hyper-phosphorylation in G(2)/M that is enhanced by IR but distinct from that on serine 272. Unlike the IR-induced phosphorylation at serine 272, this event is dependent on serine 277 and threonine 292, two C-terminal (S/T)P sites in hRad9.     

Location of the serine residue involved in the linkage between the terminal protein and the DNA of phage phi 29.

B. subtilis phage phi 29 has a terminal protein, p3, covalently linked to the 5' ends of the DNA through a phosphodiester bond between a serine residue and 5'-dAMP. This protein acts as a primer in DNA replication by forming an initiation complex with the 5'-terminal nucleotide dAMP. The amino acid sequence of the terminal protein, deduced from the nucleotide sequence of gene 3, showed the presence of 18 serine residues in a total of 266 amino acids. In this paper we have identified the serine involved in the linkage with the DNA as the residue 232, located close to the C-terminus of the molecule. This result was obtained by amino acid analysis of the peptide that remains linked to the DNA after proteinase K digestion of the terminal protein-phi 29 DNA complex and automated Edman degradation of the corresponding [125I]-labeled tryptic peptide. Prediction of the secondary structure of the terminal protein suggested that the serine residue involved in the linkage with the DNA is placed in a beta-turn, probably located on the external part of the molecule, as indicated by hydropathic values.     

Self-methylation of BspRI DNA-methyltransferase.

The DNA (cytosine-5)-methyltransferase (m5C-MTase) M.BspRI is able to accept the methyl group from the methyl donor S-adenosyl-L-methionine (AdoMet) in the absence of DNA. Transfer of the methyl group to the enzyme is a slow reaction relative to DNA methylation. Self-methylation is dependent on the native conformation of the enzyme and is inhibited by S-adenosyl-L-homocysteine, DNA and sulfhydryl reagents. Amino acid sequencing of proteolytic peptides obtained from M.BspRI, which had been methylated with [methyl-3H]AdoMet, and thin layer chromatography of the modified amino acid identified two cysteines, Cys156 and Cys181 that bind the methyl group in form of S-methylcysteine. One of the acceptor residues, Cys156 is the highly conserved cysteine which plays the role of the catalytic nucleophile of m5C-MTases.     

Scoring functions for computational algorithms applicable to the design of spiked oligonucleotides.

Protein engineering by inserting stretches of random DNA sequences into target genes in combination with adequate screening or selection methods is a versatile technique to elucidate and improve protein functions. Established compounds for generating semi-random DNA sequences are spiked oligonucleotides which are synthesised by interspersing wild type (wt) nucleotides of the target sequence with certain amounts of other nucleotides. Directed spiking strategies reduce the complexity of a library to a manageable format compared with completely random libraries. Computational algorithms render feasible the calculation of appropriate nucleotide mixtures to encode specified amino acid subpopulations. The crucial element in the ranking of spiked codons generated during an iterative algorithm is the scoring function. In this report three scoring functions are analysed: the sum-of-square-differences function s, a modified cubic function c, and a scoring function m derived from maximum likelihood considerations. The impact of these scoring functions on calculated amino acid distributions is demonstrated by an example of mutagenising a domain surrounding the active site serine of subtilisin-like proteases. At default weight settings of one for each amino acid, the new scoring function m is superior to functions s and c in finding matches to a given amino acid population.     

Genetics of serine pathway enzymes in Methylobacterium extorquens AM1: phosphoenolpyruvate carboxylase and malyl coenzyme A lyase.

Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium that uses the serine pathway for formaldehyde incorporation as its assimilation pathway during growth on one-carbon compounds. A DNA region from M. extorquens AM1 previously shown to contain genes for the serine pathway enzymes malyl coenzyme A (CoA) lyase and hydroxypyruvate reductase has been characterized in more detail. Insertion mutagenesis revealed an additional region required for growth on one-carbon compounds, and all of the insertion mutants in this region lacked activity for another serine pathway enzyme, the acetyl-CoA-independent phosphoenolpyruvate (PEP) carboxylase. Expression analysis with Escherichia coli of DNA fragments that included the malyl-CoA lyase and PEP carboxylase regions identified five polypeptides, all transcribed in the same direction. Three of these polypeptides were expressed from the region necessary for the acetyl-CoA-independent PEP carboxylase, one was expressed from the region containing the malyl-CoA lyase gene, and the fifth was expressed from a region immediately downstream from the gene encoding hydroxypyruvate reductase. All six genes are transcribed in the same direction, but the transposon insertion data suggest that they are not all cotranscribed.     

Structural basis for catalytic activation of a serine recombinase

Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 ? crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.     

Polymorphic HSRs in chromosome 1 of the two semispecies Mus musculus musculus and M. m. domesticus have a common origin in an ancestral population

HSRs (homogeneously staining regions) are the cytological correlates of DNA amplification. In the house mouse, Mus musculus, many populations are polymorphic for the presence or absence of HSRs on chromosome 1. In the semispecies M. m. domesticus the amplified DNA is present within one HSR, whereas in M. m. musculus chromosomes 1 with two HSRs are found. Hybridization of HSR-specific probes to Southern blots of HSR-carrying genomic DNAs from different localities and semispecies revealed similar complex band patterns. the remaining variation is restricted to sequences with a low degree of amplification. Variation is higher between semispecies than within one semispecies. It is assumed that HSRs are derived from one original amplification event and that unequal recombination is the mechanism underlying the length variation of HSRs present today in both semispecies. Evidence from G-banding and in situ hybridization shows that the two HSRs of M. m. musculus originated from a single HSR by means of a paracentric inversion, where one break-point was located within the single HSR and the second outside the HSR. As a consequence of the paracentric inversion the two HSRs of M. m. musculus are permanently linked together. Since exchange of genes between the two semispecies is restricted to a narrow hybrid zone the amplification that gave rise to the HSR most probably occurred prior to the divergence into the semispecies M. m. domesticus and M. m. musculus about 1 million years ago.by D. Schweizer     

Limited accessibility of DNA methylation sites for bacterial methylases M. Eco RII and M.Eco dam in chromatin at different levels of organization

The bacterial methylases M. Eco RII and M. Eco dam can methylate DNA in rat liver chromatin to form the 5-methylcytosine (m5C) and N6-methyladenine (m6A) residues, respectively. The CH3-accepting capacity of DNA in chromatin (mono- and dinucleosomes, mono- and dinucleomers) is 15 - 30 times less than that of free total DNA in rat liver. Such a low level of DNA methylation in chromatin in vitro suggests that the accessibility and recognition of methylation sites by DNA-methylases are decreased in comparison with free DNA both in the core-particle DNA and in the internucleosomal DNA. The degree of DNA methylation in chromatin particles depends on the ionic strength and Mg2+; when the former is decreased from 0.515 down to 0.176, the DNA methylation by both enzymes is increased 2-fold. An addition of Mg2+ (1 - 2 mM) decreases the CH3-accepting capacity of nucleomeric DNA, that of nucleosomal DNA remains unchanged. Thus, the accessibility of DNA for methylases is variable depending on the conformational changes of chromatin. The values of the m6A to m5C ratio for free and nucleosomal DNAs formed by methylation with a methylation of nucleomeric DNA, i. e. 1.01, 0.92 and 0.51, respectively. As Mg/4 concentration rises, the m6A/m5C ratio for nucleosomal and nucleomeric DNA is increased. It seems therefore that at different levels of organization and upon certain conformation changes the number and, probably, the nature of exposed DNA methylation sites in chromatin are different. Bacterial DNA-methylases can be used as an effective probe for a fine analysis of chromatin ultrastructure, in particular at its different functional states.