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1.  Unusual Modification of Bacteriophage Mu DNA 
Journal of Virology  1979;32(2):468-475.
Bacteriophage Mu DNA was labeled after induction in the presence of [2-3H]adenine or [8-3H]adenine. Both Mu mom+·dam+ DNA and Mu mom−·dam+ DNA have similar N6-methyladenine (MeAde) contents, as well as similar frequencies of MeAde nearest neighbors. Both DNAs are sensitive to in vitro cleavage by R·DpnI but resistant to cleavage by R·DpnII. These results indicate that the mom+ protein does not alter the sequence specificity of the host dam+ methylase to produce MeAde at new sites. However, we have discovered a new modified base, denoted Ax, in Mu mom+·dam+ DNA; approximately 15% of the adenine residues are modified to Ax. Although the precise nature of the modification is not yet defined, analysis by electrophoresis and chromatography indicates that the N6-amino group is not the site of modification, and that the added moiety contains a free carboxyl group. Ax is not present in Mu mom+·dam+ or Mu mom−·dam+ phage DNA or in cellular DNA from uninduced Mu mom+·dam+ lysogens. These results suggest that expression of the dam+ and mom+ genes are required for the Ax modification and that this modification is responsible for protecting Mu DNA against certain restriction nucleases. Mu mom+·dam− DNA and Mu mom−·dam− DNA contain a very low level of MeAde (ca. 1 MeAde per 5,000 adenine residues). Since the only nearest neighbor to MeAde appears to be cytosine, we suggest that the methylated sequence is 5′... C-A*-C... 3′ and that this methylation is mediated by the EcoK modification enzyme.
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PMCID: PMC353578  PMID: 159363
2.  Genetic Organization and Molecular Analysis of the EcoVIII Restriction-Modification System of Escherichia coli E1585-68 and Its Comparison with Isospecific Homologs 
The EcoVIII restriction-modification (R-M) system is carried by the Escherichia coli E1585-68 natural plasmid pEC156 (4,312 bp). The two genes were cloned and characterized. The G+C content of the EcoVIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or E. coli genomic DNA (50.8%). The difference suggests that there is a possibility that the EcoVIII R-M system was recently acquired by the genome. The 921-bp EcoVIII endonuclease (R · EcoVIII) gene (ecoVIIIR) encodes a 307-amino-acid protein with an Mr of 35,554. The convergently oriented EcoVIII methyltransferase (M · EcoVIII) gene (ecoVIIIM) consists of 912 bp that code for a 304-amino-acid protein with an Mr of 33,930. The exact positions of the start codon AUG were determined by protein microsequencing. Both enzymes recognize the specific palindromic sequence 5′-AAGCTT-3′. Preparations of EcoVIII R-M enzymes purified to homogeneity were characterized. R · EcoVIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5′ protruding ends. M · EcoVIII functions as a monomer and modifies the first adenine residue at the 5′ end of the specific sequence to N6-methyladenine. These enzymes are thus functionally identical to the corresponding enzymes of the HindIII (Haemophilus influenzae Rd) and LlaCI (Lactococcus lactis subsp. cremoris W15) R-M systems. This finding is reflected by the levels of homology of M · EcoVIII with M · HindIII and M · LlaCI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m6 N-adenine β-class methyltransferases. The deduced amino acid sequence of R · EcoVIII shows weak homology with its two isoschizomers, R · HindIII (26%) and R · LlaCI (17%). A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R · EcoVIII (D108X12DXK123), as well as in the primary structures of R · LlaCI and R · HindIII. Polyclonal antibodies raised against R · EcoVIII did not react with R · HindIII, while anti-M · EcoVIII antibodies cross-reacted with M · LlaCI but not with M · HindIII. R · EcoVIII requires Mg(II) ions for phosphodiester bond cleavage. We found that the same ions are strong inhibitors of the M · EcoVIII enzyme. The biological implications of this finding are discussed.
doi:10.1128/AEM.69.5.2638-2650.2003
PMCID: PMC154532  PMID: 12732532
3.  DNA Methylation 
EcoSal Plus  2014;2014:10.1128/ecosalplus.ESP-0003-2013.
The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function.
doi:10.1128/ecosalplus.ESP-0003-2013
PMCID: PMC4231299  PMID: 25405210
4.  A DNA Adenine Methyltransferase of Escherichia coli That Is Cell Cycle Regulated and Essential for Viability 
Journal of Bacteriology  2004;186(7):2061-2067.
DNA sequence analysis revealed that the putative yhdJ DNA methyltransferase gene of Escherichia coli is 55% identical to the Nostoc sp. strain PCC7120 gene encoding DNA methyltransferase AvaIII, which methylates adenine in the recognition sequence, ATGCAT. The yhdJ gene was cloned, and the enzyme was overexpressed and purified. Methylation and restriction analysis showed that the DNA methyltransferase methylates the first adenine in the sequence ATGCAT. This DNA methylation was found to be regulated during the cell cycle, and the DNA adenine methyltransferase was designated M.EcoKCcrM (for “cell cycle-regulated methyltransferase”). The CcrM DNA adenine methyltransferase is required for viability in E. coli, as a strain lacking a functional genomic copy of ccrM can be isolated only in the presence of an additional copy of ccrM supplied in trans. The cells of such a knockout strain stopped growing when expression of the inducible plasmid ccrM gene was shut off. Overexpression of M.EcoKCcrM slowed bacterial growth, and the ATGCAT sites became fully methylated throughout the cell cycle; a high proportion of cells with an anomalous size distribution and DNA content was found in this population. Thus, the temporal control of this methyltransferase may contribute to accurate cell cycle control of cell division and cellular morphology. Homologs of M.EcoKCcrM are present in other bacteria belonging to the gamma subdivision of the class Proteobacteria, suggesting that methylation at ATGCAT sites may have similar functions in other members of this group.
doi:10.1128/JB.186.7.2061-2067.2004
PMCID: PMC374390  PMID: 15028690
5.  Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus. 
Nucleic Acids Research  1996;24(14):2760-2766.
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.
PMCID: PMC146015  PMID: 8759008
6.  Antibodies Specific for the Hia Adhesion Proteins of Nontypeable Haemophilus influenzae Mediate Opsonophagocytic Activity▿  
The Hia autotransporter proteins are highly immunogenic surface adhesins expressed by nontypeable Haemophilus influenzae (NTHI). The objective of our study was to assess the opsonophagocytic activity of anti-Hia antibodies against homologous and heterologous NTHI. A segment of the hia gene that encodes a surface-exposed portion of the H. influenzae strain 11 Hia protein was cloned into a pGEMEX-2 expression vector. Escherichia coli JM101 was transformed with the resulting pGEMEX-Hia BstEII del recombinant plasmid, and recombinant fusion protein was recovered. An immune serum against recombinant GEMEX-Hia (rGEMEX-Hia)-mediated killing of the homologous NTHI strain 11 at a 1:160 titer and five heterologous Hia-expressing strains at titers of ≥1:40. Immune serum did not mediate killing of two Hia-knockout strains whose hia genes were inactivated but did mediate killing of one knockout strain at a high titer after the strain was transformed with a plasmid containing the hia gene. Immune serum did not mediate killing of HMW1/HMW2-expressing NTHI strains, which do not express the Hia adhesin. However, when two representative HMW1/HMW2-expressing strains were transformed with the plasmid containing the hia gene, they expressed abundant Hia and were susceptible to killing by the immune serum. Immune serum did not mediate killing of HMW1/HMW2-expressing strains transformed with the plasmid without the hia gene. Our results demonstrate that the Hia proteins of NTHI are targets of opsonophagocytic antibodies and that shared epitopes recognized by such antibodies are present on the Hia proteins of unrelated NTHI strains. These data argue for the continued investigation of the Hia proteins as vaccine candidates for the prevention of NTHI disease.
doi:10.1128/CVI.00090-09
PMCID: PMC2708409  PMID: 19474261
7.  Functional Analysis of MmeI from Methanol Utilizer Methylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes▿  
MmeI from Methylophilus methylotrophus belongs to the type II restriction-modification enzymes. It recognizes an asymmetric DNA sequence, 5′-TCCRAC-3′ (R indicates G or A), and cuts both strands at fixed positions downstream of the specific site. This particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). We have shown previously that the endonucleolytic activity of MmeI is strongly dependent on the presence of S-adenosyl-l-methionine (J. Nakonieczna, J. W. Zmijewski, B. Banecki, and A. J. Podhajska, Mol. Biotechnol. 37:127-135, 2007), which puts MmeI in subtype IIG. The same cofactor is used by MmeI as a methyl group donor for modification of an adenine in the upper strand of the recognition site to N6-methyladenine. Both enzymatic activities reside in a single polypeptide (919 amino acids [aa]), which puts MmeI also in subtype IIC of the restriction-modification systems. Based on a molecular model, generated with the use of bioinformatic tools and validated by site-directed mutagenesis, we were able to localize three functional domains in the structure of the MmeI enzyme: (i) the N-terminal portion containing the endonucleolytic domain with the catalytic Mg2+-binding motif D70-X9-EXK82, characteristic for the PD-(D/E)XK superfamily of nucleases; (ii) a central portion (aa 310 to 610) containing nine sequence motifs conserved among N6-adenine γ-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.
doi:10.1128/AEM.01322-08
PMCID: PMC2612229  PMID: 18997032
8.  Expression of Escherichia coli dam gene in Bacillus subtilis provokes DNA damage response: N6-methyladenine is removed by two repair pathways. 
Nucleic Acids Research  1992;20(14):3607-3615.
The dam gene of Escherichia coli encodes a DNA methyltransferase that methylates the N6 position of adenine in the sequence GATC. It was stably expressed from a shuttle vector in a repair- and recombination-proficient strain of Bacillus subtilis. In this strain the majority of plasmid DNA molecules was modified at dam sites whereas most chromosomal DNA remained unmethylated during exponential growth. During stationary phase the amount of unmethylated DNA increased, suggesting that methylated bases were being removed. An ultraviolet damage repair-deficient mutant (uvrB) contained highly methylated chromosomal and plasmid DNA. High levels of Dam methylation were detrimental to growth and viability of this mutant strain and some features of the SOS response were also induced. A mutant defective in the synthesis of adaptive DNA alkyltransferases and induction of the adaptive response (ada) also showed high methylation and properties similar to that of the dam gene expressing uvrB strain. When protein extracts from B. subtilis expressing the Dam methyltransferase or treated with N-methyl-N'-nitro-N-nitroso-guanidine were incubated with [3H]-labelled Dam methylated DNA, the methyl label was bound to two proteins of 14 and 9 kD. Some free N6-methyladenine was also detected in the supernatant of the incubation mixture. We propose that N6-methyladenine residues are excised by proteins involved in both excision (uvrB) and the adaptive response (ada) DNA repair pathways in B. subtilis.
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PMCID: PMC334008  PMID: 1641327
9.  Kinetic Analysis of Yersinia pestis DNA Adenine Methyltransferase Activity Using a Hemimethylated Molecular Break Light Oligonucleotide 
PLoS ONE  2007;2(8):e801.
Background
DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam) has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics.
Methodology/Principal Findings
Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71±0.07 indicating that it is a sensitive assay for the identification of inhibitors.
Conclusions/Significance
The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.
doi:10.1371/journal.pone.0000801
PMCID: PMC1949145  PMID: 17726531
10.  MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection 
Nucleic Acids Research  2008;36(20):6558-6570.
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.
doi:10.1093/nar/gkn711
PMCID: PMC2582602  PMID: 18931376
11.  Characterization of BseMII, a new type IV restriction–modification system, which recognizes the pentanucleotide sequence 5′-CTCAG(N)10/8↓ 
Nucleic Acids Research  2001;29(4):895-903.
We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5′-CTCAG sequence, and the nucleotide sequence of the genes encoding them. The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3′-protruding end. Magnesium ions and S-adenosyl-l-methionine (AdoMet) are required for cleavage. S-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5′-CTCAG-3′/5′-CTGAG-3′. Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5′-CTCAG strand of the target duplex. The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-l-methionine binding and catalysis. According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.
PMCID: PMC29615  PMID: 11160921
12.  Escherichia coli OxyR protein represses the unmethylated bacteriophage Mu mom operon without blocking binding of the transcriptional activator C. 
Nucleic Acids Research  1996;24(20):4042-4049.
Transcription of the bacteriophage Mu mom operon requires transactivation by the phage-encoded C protein. DNase I footprinting showed that in the absence of C, Escherichia coli RNA polymerase E(sigma)70 (RNAP) binds to the mom promoter (Pmom) region at a site, P2 (from -64 to -11 with respect to the transcription start site), on the top (non-transcribed) strand. This is slightly upstream from, but overlapping P1 (-49 to +16), the functional binding site for rightward transcription. Host DNA-[N6-adenine] methyltransferase (Dam) methylation of three GATCs immediately upstream of the C binding site is required to prevent binding of the E.coli OxyR protein, which represses mom transcription in dam- strains. OxyR, known to induce DNA bending, is normally in a reduced conformation in vivo, but is converted to an oxidized state under standard in vitro conditions. Using DNase I footprinting, we provide evidence supporting the proposal that the oxidized and reduced forms of OxyR interact differently with their target DNA sequences in vitro. A mutant form, OxyR-C199S, was shown to be able to repress mom expression in vivo in a dam- host. In vitro DNase I footprinting showed that OxyR-C199S protected Pmom from -104 to -46 on the top strand and produced a protection pattern characteristic of reduced wild-type OxyR. Prebinding of OxyR-C199S completely blocked RNAP binding to P2 (in the absence of C), whereas it only slightly decreased binding of C to its target site (-55 to -28, as defined by DNase I footprinting). In contrast, OxyR-C199S strongly inhibited C-activated recruitment of RNAP to P1. These results indicate that OxyR repression is mediated subsequent to binding by C. Mutations have been isolated that relieve the dependence on C activation and have the same transcription start site as the C-activated wild-type promoter. One such mutant, tin7, has a single base change at -14, which changes a T6 run to T3GT2. OxyR-C199S partially inhibited RNAP binding to the tin7 promoter in vitro, even though the OxyR and RNAP-P1 binding sites probably do not overlap, and in vivo expression of tin7 was reduced 5- to 10-fold in dam- cells. These results suggest that OxyR can repress tin7.
PMCID: PMC146201  PMID: 8918810
13.  Structure of RsrI methyltransferase, a member of the N6-adenine β class of DNA methyltransferases 
Nucleic Acids Research  2000;28(20):3950-3961.
DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction–modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N6-adenine methyltransferase belonging to the β class of bacterial methyltransferases is described here. The structure of M·RsrI from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 Å resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PvuII methyltransferase, a cytosine amino methyltransferase of the same β group. The larger nucleotide binding pocket observed in M·RsrI is expected because it methylates adenine. However, the most striking difference between the RsrI methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.
PMCID: PMC110776  PMID: 11024175
14.  Purification, cloning and sequence analysis of RsrI DNA methyltransferase: lack of homology between two enzymes, RsrI and EcoRI, that methylate the same nucleotide in identical recognition sequences. 
Nucleic Acids Research  1989;17(24):10403-10425.
RsrI DNA methyltransferase (M-RsrI) from Rhodobacter sphaeroides has been purified to homogeneity, and its gene cloned and sequenced. This enzyme catalyzes methylation of the same central adenine residue in the duplex recognition sequence d(GAATTC) as does M-EcoRI. The reduced and denatured molecular weight of the RsrI methyltransferase (MTase) is 33,600 Da. A fragment of R. sphaeroides chromosomal DNA exhibited M.RsrI activity in E. coli and was used to sequence the rsrIM gene. The deduced amino acid sequence of M.RsrI shows partial homology to those of the type II adenine MTases HinfI and DpnA and N4-cytosine MTases BamHI and PvuII, and to the type III adenine MTases EcoP1 and EcoP15. In contrast to their corresponding isoschizomeric endonucleases, the deduced amino acid sequences of the RsrI and EcoRI MTases show very little homology. Either the EcoRI and RsrI restriction-modification systems assembled independently from closely related endonuclease and more distantly related MTase genes, or the MTase genes diverged more than their partner endonuclease genes. The rsrIM gene sequence has also been determined by Stephenson and Greene (Nucl. Acids Res. (1989) 17, this issue).
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PMCID: PMC335309  PMID: 2690017
15.  The Type ISP Restriction–Modification enzymes LlaBIII and LlaGI use a translocation–collision mechanism to cleave non-specific DNA distant from their recognition sites 
Nucleic Acids Research  2012;41(2):1071-1080.
The Type ISP Restriction–Modification (RM) enzyme LlaBIII is encoded on plasmid pJW566 and can protect Lactococcus lactis strains against bacteriophage infections in milk fermentations. It is a single polypeptide RM enzyme comprising Mrr endonuclease, DNA helicase, adenine methyltransferase and target-recognition domains. LlaBIII shares >95% amino acid sequence homology across its first three protein domains with the Type ISP enzyme LlaGI. Here, we determine the recognition sequence of LlaBIII (5′-TnAGCC-3′, where the adenine complementary to the underlined base is methylated), and characterize its enzyme activities. LlaBIII shares key enzymatic features with LlaGI; namely, adenosine triphosphate-dependent DNA translocation (∼309 bp/s at 25°C) and a requirement for DNA cleavage of two recognition sites in an inverted head-to-head repeat. However, LlaBIII requires K+ ions to prevent non-specific DNA cleavage, conditions which affect the translocation and cleavage properties of LlaGI. By identifying the locations of the non-specific dsDNA breaks introduced by LlaGI or LlaBIII under different buffer conditions, we validate that the Type ISP RM enzymes use a common translocation–collision mechanism to trigger endonuclease activity. In their favoured in vitro buffer, both LlaGI and LlaBIII produce a normal distribution of random cleavage loci centred midway between the sites. In contrast, LlaGI in K+ ions produces a far more distributive cleavage profile.
doi:10.1093/nar/gks1209
PMCID: PMC3553950  PMID: 23222132
16.  Insensitivity of chloroplast gene expression to DNA methylation 
Molecular Genetics and Genomics   2009;282(1):17-24.
Presence and possible functions of DNA methylation in plastid genomes of higher plants have been highly controversial. While a number of studies presented evidence for the occurrence of both cytosine and adenine methylation in plastid genomes and proposed a role of cytosine methylation in the transcriptional regulation of plastid genes, several recent studies suggested that at least cytosine methylation may be absent from higher plant plastid genomes. To test if either adenine or cytosine methylation can play a regulatory role in plastid gene expression, we have introduced cyanobacterial genes for adenine and cytosine DNA methyltransferases (methylases) into the tobacco plastid genome by chloroplast transformation. Using DNA cleavage with methylation-sensitive and methylation-dependent restriction endonucleases, we show that the plastid genomes in the transplastomic plants are efficiently methylated. All transplastomic lines are phenotypically indistinguishable from wild-type plants and, moreover, show no alterations in plastid gene expression. Our data indicate that the expression of plastid genes is not sensitive to DNA methylation and, hence, suggest that DNA methylation is unlikely to be involved in the transcriptional regulation of plastid gene expression.
doi:10.1007/s00438-009-0440-z
PMCID: PMC2695549  PMID: 19290543
Chloroplast; Adenine methylation; Cytosine methylation; Dam methylation; Plastid transformation; Nicotiana tabacum
17.  Multiple interfaces between a serine recombinase and an enhancer control site-specific DNA inversion 
eLife  2013;2:e01211.
Serine recombinases are often tightly controlled by elaborate, topologically-defined, nucleoprotein complexes. Hin is a member of the DNA invertase subclass of serine recombinases that are regulated by a remote recombinational enhancer element containing two binding sites for the protein Fis. Two Hin dimers bound to specific recombination sites associate with the Fis-bound enhancer by DNA looping where they are remodeled into a synaptic tetramer competent for DNA chemistry and exchange. Here we show that the flexible beta-hairpin arms of the Fis dimers contact the DNA binding domain of one subunit of each Hin dimer. These contacts sandwich the Hin dimers to promote remodeling into the tetramer. A basic region on the Hin catalytic domain then contacts enhancer DNA to complete assembly of the active Hin tetramer. Our results reveal how the enhancer generates the recombination complex that specifies DNA inversion and regulates DNA exchange by the subunit rotation mechanism.
DOI: http://dx.doi.org/10.7554/eLife.01211.001
eLife digest
Many processes in biology rely on enzymes that break both the strands in a DNA molecule, then rearrange the strands, and finally join them back together in a new configuration. These recombination reactions can, for example, change the positions of genetic elements such as enhancers and promoters within the DNA molecule and, therefore, influence how a given gene is expressed as a protein. Cells need to be able to control recombination reactions because they can lead to leukemia and lymphomas if they go wrong.
The enzymes that catalyze these recombination reactions are called recombinases. One type of recombinase binds to specific sequences of DNA bases and uses an amino acid in the enzyme–usually serine or tyrosine–to break and rejoin the DNA strands. Recombination reactions require the assembly of complexes containing many proteins bound to DNA. Tyrosine recombinases form relatively simple protein-DNA complexes, and these have been studied in detail. Serine recombinases, on the other hand, form more elaborate protein-DNA complexes, and much less is known about these.
Now McLean et al. have unraveled the mechanism that a serine recombinase called Hin uses to reverse the direction of a stretch of chromosomal DNA in the bacteria Salmonella enterica. Inverting this stretch of DNA–which contains about 1000 base pairs–changes the position of a gene promoter that is responsible for the production of flagellin, which is the protein that enables the bacterium to move. This is one of the tricks that Salmonella uses to evade the immune system of its host.
Previous research has established that four Hin subunits and two copies of a protein called Fis are needed to invert this stretch of DNA: two Hin subunits bind to each of the two hix recombination sites, and the Fis proteins (which are dimers) bind to each end of an enhancer that is located between the hix sites. A protein called HU then causes the DNA to bend and form a loop, and the four Hin subunits and the two Fis dimers all come together at the enhancer to form a structure called the invertasome where the recombination reaction occurs. All four DNA strands at the crossover point are broken as a result of a near simultaneous attack by the catalytic serine amino acids in the Hin subunits. One pair of Hin subunits–and the two DNA strands attached to them–then rotate by 180 degrees around the other pair of Hin subunits. This means that the stretch of DNA between the hix sites is inverted when the DNA strands are rejoined at the end of the reaction.
Enhancers often regulate transcription and other reactions from a distance. McLean et al. reveal how an enhancer of a DNA recombination reaction works. The pairs of Hin subunits that initially bind to the DNA are not catalytically active, but when they are brought together by the enhancer and form a tetramer, they become active. Two of the Hin subunits are clamped onto the enhancer by the Fis dimers and by directly interacting with the enhancer DNA, but the other two (and the DNA strands attached to them) are free to rotate within the tetramer. In the Salmonella chromosome the enhancer is located close to one of the hix sites (∼100 base pairs away from it), so the length of the DNA between the enhancer and hix site physically limits the number of Hin subunit rotations to just one.
DOI: http://dx.doi.org/10.7554/eLife.01211.002
doi:10.7554/eLife.01211
PMCID: PMC3798978  PMID: 24151546
Salmonella enterica; site-specific DNA recombination; serine recombinase; recombinational enhancer; synaptic complex; DNA strand exchange; E. coli
18.  In vivo specificity of EcoRI DNA methyltransferase. 
Nucleic Acids Research  1992;20(22):6091-6096.
The EcoRI adenine DNA methyltransferase forms part of a bacterial restriction/modification system; the methyltransferase modifies the second adenine within the canonical site GAATTC, thereby preventing the EcoRI endonuclease from cleaving this site. We show that five noncanonical EcoRI sites (TAATTC, CAATTC, GTATTC, GGATTC and GAGTTC) are not methylated in vivo under conditions when the canonical site is methylated. Only when the methyltransferase is overexpressed is partial in vivo methylation of the five sites detected. Our results suggest that the methyltransferase does not protect host DNA against potential endonuclease-mediated cleavage at noncanonical sites. Our related in vitro analysis of the methyltransferase reveals a low level of sequence-discrimination. We propose that the high in vivo specificity may be due to the active removal of methylated sequences by DNA repair enzymes (J. Bacteriology (1987), 169 3243-3250).
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PMCID: PMC334477  PMID: 1461739
19.  Adenine methylation may contribute to endosymbiont selection in a clonal aphid population 
BMC Genomics  2014;15(1):999.
Background
The pea aphid Acyrthosiphon pisum has two modes of reproduction: parthenogenetic during the spring and summer and sexual in autumn. This ability to alternate between reproductive modes and the emergence of clonal populations under favorable conditions make this organism an interesting model for genetic and epigenetic studies. The pea aphid hosts different types of endosymbiotic bacteria within bacteriocytes which help the aphids survive and adapt to new environmental conditions and habitats. The obligate endosymbiont Buchnera aphidicola has a drastically reduced and stable genome, whereas facultative endosymbionts such as Regiella insecticola have large and dynamic genomes due to phages, mobile elements and high levels of genetic recombination. In previous work, selection toward cold adaptation resulted in the appearance of parthenogenetic A. pisum individuals characterized by heavier weights and remarkable green pigmentation.
Results
Six adenine-methylated DNA fragments were isolated from genomic DNA (gDNA) extracted from the cold-induced green variant of A. pisum using deoxyadenosine methylase (Dam) by digesting the gDNA with the restriction enzymes DpnI and DpnII, which recognize the methylated and unmethylated GATC sites, respectively. The six resultant fragments did not match any sequence in the A. pisum or Buchnera genomes, implying that they came from facultative endosymbionts. The A1 fragment encoding a putative transposase and the A6 fragment encoding a putative helicase were selected for further comparison between the two A. pisum variants (green and orange) based on Dam analysis followed by PCR amplification. An association between adenine methylation and the two A. pisum variants was demonstrated by higher adenine methylation levels on both genes in the green variant as compared to the orange one.
Conclusion
Temperature selection may affect the secondary endosymbiont and the sensitive Dam involved in the survival and adaptation of aphids to cold temperatures. There is a high degree of adenine methylation at the GATC sites of the endosymbiont genes at 8°C, an effect that disappears at 22°C. We suggest that endosymbionts can be modified or selected to increase host fitness under unfavorable climatic conditions, and that the phenotype of the newly adapted aphids can be inherited.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-999) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-999
PMCID: PMC4246565  PMID: 25406741
Acyrthosiphon pisum; Bacteriocyte; Facultative/secondary bacterium; Epigenetic; Deoxyadenosine methylase (Dam)
20.  A genome-wide screen for modifiers of transgene variegation identifies genes with critical roles in development 
Genome Biology  2008;9(12):R182.
An extended ENU screen for modifiers of transgene variegation identified four new modifiers, MommeD7-D10.
Background
Some years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design.
Results
We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome.
Conclusions
These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.
doi:10.1186/gb-2008-9-12-r182
PMCID: PMC2646286  PMID: 19099580
21.  Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695 
PLoS ONE  2011;6(2):e16810.
HP0593 DNA-(N6-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5′-GCAG-3′and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co2+, Mn2+, and also Mg2+ stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.
doi:10.1371/journal.pone.0016810
PMCID: PMC3036652  PMID: 21347417
22.  Methylation of ribosomal RNA genes in the macronucleus of Tetrahymena thermophila. 
Nucleic Acids Research  1983;11(15):5131-5145.
We have investigated the occurrence of methylated adenine residues in the macronuclear ribosomal RNA genes of Tetrahymena thermophila. It has been shown previously that macronuclear DNA, including the palindromic ribosomal RNA genes (rDNA), of Tetrahymena thermophila contains the modified base N-6-methyladenine, but no 5-methylcytosine. Purified rDNA was digested with restriction enzymes Sau 3AI, MboI and DpnI to map the positions and levels of N-6-methyladenine in the sequence 5' GATC 3'. A specific pattern of doubly methylated GATC sequences was found; hemimethylated sites were not detected. The patterns and levels of methylation of these sites did not change significantly in different physiological states. A molecular form of the rDNA found in the newly developing macronucleus and for several generations following the sexual process, conjugation, contained no detectably methylated GATC sites. However, both the bulk macronuclear DNA and palindromic rDNA from the same macronuclei were methylated. Possible roles for N-6-methyladenine in macronuclear DNA are discussed in light of these findings.
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PMCID: PMC326242  PMID: 6308573
23.  DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila  
Infection and Immunity  2006;74(1):410-424.
Among the various virulence factors produced by Aeromonas hydrophila, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of Aeromonas-associated infections. Our laboratory molecularly characterized both Act and the T3SS from a diarrheal isolate, SSU of A. hydrophila, and defined the role of some regulatory genes in modulating the biological effects of Act. In this study, we cloned, sequenced, and expressed the DNA adenine methyltransferase gene of A. hydrophila SSU (damAhSSU) in a T7 promoter-based vector system using Escherichia coli ER2566 as a host strain, which could alter the virulence potential of A. hydrophila. Recombinant Dam, designated as M.AhySSUDam, was produced as a histidine-tagged fusion protein and purified from an E. coli cell lysate using nickel affinity chromatography. The purified Dam had methyltransferase activity, based on its ability to transfer a methyl group from S-adenosyl-l-methionine to N6-methyladenine-free lambda DNA and to protect methylated lambda DNA from digestion with DpnII but not against the DpnI restriction enzyme. The dam gene was essential for the viability of the bacterium, and overproduction of Dam in A. hydrophila SSU, using an arabinose-inducible, PBAD promoter-based system, reduced the virulence of this pathogen. Specifically, overproduction of M.AhySSUDam decreased the motility of the bacterium by 58%. Likewise, the T3SS-associated cytotoxicity, as measured by the release of lactate dehydrogenase enzyme in murine macrophages infected with the Dam-overproducing strain, was diminished by 55% compared to that of a control A. hydrophila SSU strain harboring the pBAD vector alone. On the contrary, cytotoxic and hemolytic activities associated with Act as well as the protease activity in the culture supernatant of a Dam-overproducing strain were increased by 10-, 3-, and 2.4-fold, respectively, compared to those of the control A. hydrophila SSU strain. The Dam-overproducing strain was not lethal to mice (100% survival) when given by the intraperitoneal route at a dose twice that of the 50% lethal dose, which within 2 to 3 days killed 100% of the animals inoculated with the A. hydrophila control strain. Taken together, our data indicated alteration of A. hydrophila virulence by overproduction of Dam.
doi:10.1128/IAI.74.1.410-424.2006
PMCID: PMC1346675  PMID: 16368997
24.  Construction and Immunogenicity of Recombinant Adenovirus Vaccines Expressing the HMW1, HMW2, or Hia Adhesion Protein of Nontypeable Haemophilus influenzae▿  
Clinical and Vaccine Immunology : CVI  2010;17(10):1567-1575.
The objective of the present study was to construct and assess the immunogenicity of recombinant adenovirus vectors expressing the HMW1, HMW2, or Hia protein of nontypeable Haemophilus influenzae (NTHi). These proteins are critical adhesins and potential protective antigens expressed by NTHi. Segments of the hmw1A and hmw2A structural genes that encode the distal one-half of mature HMW1 or HMW2 were cloned into the T7 expression vector pGEMEX-2. These constructs encoded stable HMW1 or HMW2 recombinant fusion protein that expresses B-cell epitopes common to most NTHi strains. A segment of the hia gene that encodes the surface-exposed portion of mature Hia was also cloned into pGEMEX-2. The resulting T7 gene 10 translational fusions were excised from the parent plasmids and cloned into the shuttle plasmid pDC316. Cotransfection of HEK 293 cells with the pDC316 derivatives and pBHGloxΔE1,3Cre resulted in the production of viral plaques from which recombinant adenoviruses expressing fusion proteins were recovered. Chinchillas immunized intraperitoneally with a single 108-PFU dose of either the HMW2 or Hia adenoviral construct developed high anti-HMW2 or anti-Hia serum antibody titers within 4 weeks of immunization. Chinchillas immunized intranasally with a single 107- to 109-PFU dose of the Hia adenoviral construct also developed high anti-Hia serum antibody titers within 8 weeks of immunization. Recombinant adenoviruses represent a promising system to induce mucosal and systemic immunity and protection against mucosal diseases such as otitis media. Recombinant adenoviruses expressing recombinant HMW1, HMW2, or Hia protein will be important new tools in NTHi vaccine development efforts.
doi:10.1128/CVI.00115-10
PMCID: PMC2952983  PMID: 20685934
25.  DNA cleavage and methylation specificity of the single polypeptide restriction–modification enzyme LlaGI 
Nucleic Acids Research  2009;37(21):7206-7218.
LlaGI is a single polypeptide restriction–modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a γ-family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5′-CTnGAyG-3′ (where n is A, G, C or T and y is C or T). Methylation of the recognition site occurred on only one strand (the non-degenerate dA residue of 5′-CrTCnAG-3′ being methylated at the N6 position). Double strand DNA breaks at distant, random sites were only observed when two head-to-head oriented, unmethylated copies of the site were present; single sites or pairs in tail-to-tail or head-to-tail repeat only supported a DNA nicking activity. dsDNA nuclease activity was dependent upon the presence of ATP or dATP. Our results are consistent with a directional long-range communication mechanism that is necessitated by the partial site methylation. In the accompanying manuscript [Smith et al. (2009) The single polypeptide restriction–modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops], we demonstrate that this communication is via 1-dimensional DNA loop translocation. On the basis of this data and that in the third accompanying manuscript [Smith et al. (2009) An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI], we propose that LlaGI is the prototype of a new sub-classification of Restriction-Modification enzymes, named Type I SP (for Single Polypeptide).
doi:10.1093/nar/gkp790
PMCID: PMC2790903  PMID: 19808936

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