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1.  Real time kinetics of restriction endonuclease cleavage monitored by fluorescence resonance energy transfer. 
Nucleic Acids Research  1994;22(15):3155-3159.
The kinetics of PaeR7 endonuclease-catalysed cleavage reactions of fluorophor-labeled oligonucleotide substrates have been examined using fluorescence resonance energy transfer (FRET). A series of duplex substrates were synthesized with an internal CTCGAG PaeR7 recognition site and donor (fluorescein) and acceptor (rhodamine) dyes conjugated to the opposing 5' termini. The time-dependent increase in donor fluorescence resulting from restriction cleavage of these substrates was continuously monitored and the initial rate data was fitted to the Michaelis-Menten equation. The steady state kinetic parameters for these substrates were in agreement with the rate constants obtained from a gel electrophoresis-based fixed time point assay using radiolabeled substrates. The FRET method provides a rapid continuous assay as well as high sensitivity and reproducibility. These features should make the technique useful for the study of DNA-cleaving enzymes.
PMCID: PMC310290  PMID: 8065930
2.  Introduction and expression of the bacterial PaeR7 restriction endonuclease gene in mouse cells containing the PaeR7 methylase. 
Nucleic Acids Research  1988;16(24):11489-11506.
To study the factors essential for a functional restriction system, the PaeR7 restriction-modification system has been introduced and expressed in murine cells. Transfer of this system was accomplished in two steps. First, cells containing sufficient PaeR7 methylase to completely methylate the mouse genome were constructed. In the second step, the mouse metallothionein promoter-regulated, endonuclease expression vector linked to the hygromycin B resistance selection marker was used to transfect the high methylase-expressing cells. Sixty percent of the clones isolated contained PaeR7 endonuclease enzymatic activity. Transfected cells expressing both methylase and endonuclease were incapable of blocking infection by DNA viruses, and possible explanations are discussed.
PMCID: PMC339060  PMID: 2850539
3.  Persistence of recombinant adenovirus in vivo is not dependent on vector DNA replication. 
Journal of Virology  1997;71(11):8902-8907.
Recombinant adenovirus vectors represent an efficient means of transferring genes into many different organs. The first-generation E1-deleted vector genome remains episomal and, in the absence of host immunity, persists long-term in quiescent tissues such as the liver. The mechanism(s) which allows for persistence has not been established; however, vector DNA replication may be important because replication has been shown to occur in tissue culture systems. We have utilized a site-specific methylation strategy to monitor the replicative fate of E1-deleted adenovirus vectors in vitro and in vivo. Methylation-marked adenovirus vectors were produced by the addition of a methyl group onto the N6 position of the adenine base of XhoI sites, CTCGAG, by propagation of vectors in 293 cells expressing the XhoI isoschizomer PaeR7 methyltransferase. The methylation did not affect vector production or transgene expression but did prevent cleavage by XhoI. Loss of methylation through viral replication restores XhoI cleavage and was observed by Southern analysis in a wide variety of, but not all, cell culture systems studied, including hepatoma and mouse and macaque primary hepatocyte cultures. In contrast, following liver-directed gene transfer of methylated vector in C57BL/6 mice, adenovirus vector DNA was not cleaved by XhoI and therefore did not replicate, even after a period of 3 weeks. Although replication may occur in some tissues, these results show that stabilization of the vector within the target tissue prior to clearance by host immunity is not dependent upon replication of the vector, demonstrating that the input transduced DNA genomes were the persistent molecules. This information will be useful for the design of optimal adenovirus vectors and perhaps nonviral episomal vectors for clinical gene therapy.
PMCID: PMC192362  PMID: 9343256
4.  Cloning and sequence analysis of the genes coding for Eco57I type IV restriction-modification enzymes. 
Nucleic Acids Research  1992;20(22):6051-6056.
A 6.3 kb fragment of E.coli RFL57 DNA coding for the type IV restriction-modification system Eco57I was cloned and expressed in E.coli RR1. A 5775 bp region of the cloned fragment was sequenced which contains three open reading frames (ORF). The methylase gene is 1623 bp long, corresponding to a protein of 543 amino acids (62 kDa); the endonuclease gene is 2991 bp in length (997 amino acids, 117 kDa). The two genes are transcribed convergently from different strands with their 3'-ends separated by 69 bp. The third short open reading frame (186 bp, 62 amino acids) has been identified, that precedes and overlaps by 7 nucleotides the ORF encoding the methylase. Comparison of the deduced Eco57I endonuclease and methylase amino acid sequences revealed three regions of significant similarity. Two of them resemble the conserved sequence motifs characteristic of the DNA[adenine-N6] methylases. The third one shares similarity with corresponding regions of the PaeR7I, TaqI, CviBIII, PstI, BamHI and HincII methylases. Homologs of this sequence are also found within the sequences of the PaeR7I, PstI and BamHI restriction endonucleases. This is the first example of a family of cognate restriction endonucleases and methylases sharing homologous regions. Analysis of the structural relationship suggests that the type IV enzymes represent an intermediate in the evolutionary pathway between the type III and type II enzymes.
PMCID: PMC334472  PMID: 1334261
5.  Nucleotide sequence of the PaeR7 restriction/modification system and partial characterization of its protein products. 
Nucleic Acids Research  1985;13(23):8441-8461.
Bal31 deletion experiments on clones of the PaeR7 restriction-modification system from Pseudomonas aeruginosa demonstrate that it is arranged as an operon, with the methylase gene preceding the endonuclease gene. The DNA sequence of this operon agrees with in vitro transcription-translation assays which predict proteins of 532 amino acids, Mr = 59,260 daltons, and 246 amino acids, Mr = 27,280 daltons, coincident with the methylase and endonuclease genes, respectively. These predicted values coincide with the measured molecular weights of the purified, denatured PaeR7 endonuclease and methylase proteins. The first twenty amino acids from the amino-terminus of the purified endonuclease exactly match those predicted from the DNA sequence. Finally, potential regulatory mechanisms for the expression of phage restriction are described based on the properties of several PaeR7 subclones.
PMCID: PMC322144  PMID: 3001639
6.  Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation 
Nucleic Acids Research  2012;40(11):4841-4849.
Cytosine residues in mammalian DNA occur in at least three forms, cytosine (C), 5-methylcytosine (M; 5mC) and 5-hydroxymethylcytosine (H; 5hmC). During semi-conservative DNA replication, hemi-methylated (M/C) and hemi-hydroxymethylated (H/C) CpG dinucleotides are transiently generated, where only the parental strand is modified and the daughter strand contains native cytosine. Here, we explore the role of DNA methyltransferases (DNMT) and ten eleven translocation (Tet) proteins in perpetuating these states after replication, and the molecular basis of their recognition by methyl-CpG-binding domain (MBD) proteins. Using recombinant proteins and modified double-stranded deoxyoligonucleotides, we show that DNMT1 prefers a hemi-methylated (M/C) substrate (by a factor of >60) over hemi-hydroxymethylated (H/C) and unmodified (C/C) sites, whereas both DNMT3A and DNMT3B have approximately equal activity on all three substrates (C/C, M/C and H/C). Binding of MBD proteins to methylated DNA inhibited Tet1 activity, suggesting that MBD binding may also play a role in regulating the levels of 5hmC. All five MBD proteins generally have reduced binding affinity for 5hmC relative to 5mC in the fully modified context (H/M versus M/M), though their relative abilities to distinguish the two varied considerably. We further show that the deamination product of 5hmC could be excised by thymine DNA glycosylase and MBD4 glycosylases regardless of context.
PMCID: PMC3367191  PMID: 22362737
7.  Recruitment of Single-Stranded Recombinant Adeno-Associated Virus Vector Genomes and Intermolecular Recombination Are Responsible for Stable Transduction of Liver In Vivo 
Journal of Virology  2000;74(20):9451-9463.
Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.
PMCID: PMC112374  PMID: 11000214
8.  PaeX, a Second Pectin Acetylesterase of Erwinia chrysanthemi 3937 
Journal of Bacteriology  2003;185(10):3091-3100.
Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. Pectin is a complex polysaccharide. The main chain is constituted of galacturonate residues, and some of them are modified by methyl and/or acetyl esterification. Esterases are necessary to remove these modifications and, thus, to facilitate the further degradation of the polysaccharidic chain. In addition to PaeY, the first pectin acetylesterase identified in the E. chrysanthemi strain 3937, we showed that this bacterium produces a second pectin acetylesterase encoded by the gene paeX. The paeX open reading frame encodes a 322-residue precursor protein of 34,940 Da, including a 21-amino-acid signal peptide. Analysis of paeX transcription, by using gene fusions, revealed that it is induced by pectic catabolic products and affected by catabolite repression. The expression of paeX is regulated by the repressor KdgR, which controls all the steps of pectin catabolism; by the repressor PecS, which controls most of the pectinase genes; and by catabolite regulatory protein, the global activator of sugar catabolism. The paeX gene is situated in a cluster of genes involved in the catabolism and transport of pectic oligomers. In induced conditions, the two contiguous genes kdgM, encoding an oligogalacturonate-specific porin, and paeX are both transcribed as an operon from a promoter proximal to kdgM, but transcription of paeX can also be uncoupled from that of kdgM in noninduced conditions. PaeX is homologous to the C-terminal domain of the Butyrivibrio fibriosolvens xylanase XynB and to a few bacterial esterases. PaeX contains the typical box (GxSxG) corresponding to the active site of the large family of serine hydrolases. Purified PaeX releases acetate from various synthetic substrates and from sugar beet pectin. The PaeX activity increased after previous depolymerization and demethylation of pectin, indicating that its preferred substrates are nonmethylated oligogalacturonides. PaeX is mostly found in the periplasmic space of E. chrysanthemi. These data suggest that PaeX is mainly involved in the deacetylation of esterified oligogalacturonides that enter the periplasm by the KdgM porin.
PMCID: PMC154074  PMID: 12730169
9.  From damaged genome to cell surface: transcriptome changes during bacterial cell death triggered by loss of a restriction–modification gene complex 
Nucleic Acids Research  2009;37(9):3021-3031.
Genetically programmed cell deaths play important roles in unicellular prokaryotes. In postsegregational killing, loss of a gene complex from a cell leads to its descendants’ deaths. With type II restriction–modification gene complexes, such death is triggered by restriction endonuclease's attacks on under-methylated chromosomes. Here, we examined how the Escherichia coli transcriptome changes after loss of PaeR7I gene complex. At earlier time points, activation of SOS genes and σE-regulon was noticeable. With time, more SOS genes, stress-response genes (including σS-regulon, osmotic-, oxidative- and periplasmic-stress genes), biofilm-related genes, and many hitherto uncharacterized genes were induced, and genes for energy metabolism, motility and outer membrane biogenesis were repressed. As expected from the activation of σE-regulon, the death was accompanied by cell lysis and release of cellular proteins. Expression of several σE-regulon genes indeed led to cell lysis. We hypothesize that some signal was transduced, among multiple genes involved, from the damaged genome to the cell surface and led to its disintegration. These results are discussed in comparison with other forms of programmed deaths in bacteria and eukaryotes.
PMCID: PMC2685091  PMID: 19304752
10.  Genome typing of mouse adenoviruses. 
Restriction endonuclease cleavage site analysis was used to differentiate between mouse adenovirus (MAV) types 1 and 2 strains. Viral DNA of suitable purity and quantity for multiple enzymatic digestions was obtained from cloned CMT-93 mouse tumor cells infected with each type of MAV. Clear differences between the MAV-1 (FL) and MAV-2 (K87) genomes were observed after cleavage with restriction enzymes such as BglII, EcoRI, and PaeR7. Fast electrophoresis of DNA fragments in miniature agarose slab gels allowed rapid and unequivocal identification of the MAV strains. This relatively simple and accurate method should be quite useful to determine the different modes of transmission of mouse adenoviruses and their presence in various animal populations.
PMCID: PMC266173  PMID: 2830299
11.  Cloning, Expression, and Purification of a Thermostable Nonhomodimeric Restriction Enzyme, BslI 
Journal of Bacteriology  2000;182(4):949-955.
BslI is a thermostable type II restriction endonuclease with interrupted recognition sequence CCNNNNN/NNGG (/, cleavage position). The BslI restriction-modification system from Bacillus species was cloned and expressed in Escherichia coli. The system is encoded by three genes: the 2,739-bp BslI methylase gene (bslIM), the bslIRα gene, and the bslIRβ gene. The α and β subunits of BslI can be expressed independently in E. coli in the absence of BslI methylase (M.BslI) protection. BslI endonuclease activity can be reconstituted in vitro by mixing the two subunits together. Gel filtration chromatography and native polyacrylamide gel electrophoresis indicated that BslI forms heterodimers (αβ), heterotetramers (α2β2), and possibly oligomers in solution. Two β subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramer BslI complex (α2β2). In DNA mobility shift assays, neither subunit alone can bind DNA. DNA mobility shift activity was detected after mixing the two subunits together. Because of the symmetric recognition sequence of the BslI endonuclease, we propose that its active form is α2β2. M.BslI contains nine conserved motifs of N-4 cytosine DNA methylases within the β group of aminomethyltransferase. Synthetic duplex deoxyoligonucleotides containing cytosine hemimethylated or fully methylated at N-4 in BslI sites in the first or second cytosine are resistant to BslI digestion. C-5 methylation of the second cytosine on both strands within the recognition sequence also renders the site refractory to BslI digestion. Two putative zinc fingers are found in the α subunit of BslI endonuclease.
PMCID: PMC94369  PMID: 10648519
12.  Examination of the DNA substrate selectivity of DNA cytosine methyltransferases using mass tagging 
Nucleic Acids Research  2000;28(18):3594-3599.
The biological significance of cytosine methylation is as yet incompletely understood, but substantial and growing evidence strongly suggests that perturbation of methylation patterns, resulting from the infidelity of DNA cytosine methyltransferase, is an important component of the development of human cancer. We have developed a novel in vitro assay that allows us to quantitatively determine the DNA substrate preferences of cytosine methylases. This approach, which we call mass tagging, involves the labeling of target cytosine residues in synthetic DNA duplexes with stable isotopes, such as 15N. Methylation is then measured by the formation of 5-methylcytosine (5mC) by gas chromatography/mass spectrometry. The DNA substrate selectivity is determined from the mass spectrum of the product 5mC. With the non-symmetrical duplex DNA substrate examined in this study we find that the bacterial methyltransferase HpaII (duplex DNA recognition sequence CCGG) methylates the one methylatable cytosine of each strand similarly. Introduction of an A-C mispair at the methylation site shifts methylation exclusively to the mispaired cytosine residue. In direct competition assays with HpaII methylase we observe that the mispaired substrate is methylated more extensively than the fully complementary, normal substrate, although both have one HpaII methylation site. Through the use of this approach we will be able to learn more about the mechanisms by which methylation patterns can become altered.
PMCID: PMC110732  PMID: 10982881
13.  Substrate specificity of new methyl-directed DNA endonuclease GlaI 
Recently, we have discovered site-specific endonucleases, which recognize and cleave only DNA sequences with 5-methylcytosine. Two specificities of such endonucleases have been described. Enzymes BisI, BlsI, and GluI are isoschizomers and hydrolyze the DNA sequence 5'-GCNGC-3'/3'-CGNCG-5', which is methylated in different ways. The enzyme GlaI cleaves the DNA sequence 5'-GCGC-3'/3'-CGCG-5' if there are two, three or four 5-methylcytosines. The goal of the present work is to study in detail the composition of recognition sequence and effect of the methylated cytosines on the efficiency of DNA cleavage by the methyl-directed DNA endonuclease GlaI
In a recent work we have studied the dependence of GlaI activity on the quantity and location of 5-methylcytosines in the enzyme recognition sequence 5'-GCGC-3'/3'-CGCG-5'. A significant DNA cleavage has been observed for oligonucleotide duplexes, which include either three or four 5-methylcytosines. In this work we have studied dependence of the GlaI activity on quantity and location of methylated cytosines, as well as on composition of the recognition sequence.
The list of good substrates for GlaI includes a fully methylated site 5'-CGCG-3'/3'-GCGC-5', sites with three cytosines of a general structure 5'-PuMGM-3'/3'-PyGMG-5', and one recognition sequence with two methylated cytosines 5'-AMGT-3'/3'-TGMA-5', where M is 5-methylcytosine.
GlaI intermediate substrates include sites with three methylated cytosines of a general structure 5'-GMPuM-3'/3'-MGPyG-5', as well as a site with two methylcytosines 5'-GMGT-3'/3'-CGMA-5'.
The site 5'-GMGC-3'/3'-CGMG-5' may be considered a low activity substrate.
PMCID: PMC2257971  PMID: 18194583
14.  Site-specific methylases induce the SOS DNA repair response in Escherichia coli. 
Journal of Bacteriology  1987;169(7):3243-3250.
Expression of the site-specific adenine methylase HhaII (GmeANTC, where me is methyl) or PstI (CTGCmeAG) induced the SOS DNA repair response in Escherichia coli. In contrast, expression of methylases indigenous to E. coli either did not induce SOS (EcoRI [GAmeATTC] or induced SOS to a lesser extent (dam [GmeATC]). Recognition of adenine-methylated DNA required the product of a previously undescribed gene, which we named mrr (methylated adenine recognition and restriction). We suggest that mrr encodes an endonuclease that cleaves DNA containing N6-methyladenine and that DNA double-strand breaks induce the SOS response. Cytosine methylases foreign to E. coli (MspI [meCCGG], HaeIII [GGmeCC], BamHI [GGATmeCC], HhaI [GmeCGC], BsuRI [GGmeCC], and M.Spr) also induced SOS, whereas one indigenous to E. coli (EcoRII [CmeCA/TGG]) did not. SOS induction by cytosine methylation required the rglB locus, which encodes an endonuclease that cleaves DNA containing 5-hydroxymethyl- or 5-methylcytosine (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986).
PMCID: PMC212376  PMID: 3036779
15.  Alpha-1 Antitrypsin Reduces Severity of Pseudomonas Pneumonia in Mice and Inhibits Epithelial Barrier Disruption and Pseudomonas Invasion of Respiratory Epithelial Cells 
Nosocomial pneumonia (NP) is the third most common hospital-acquired infection and the leading cause of death due to hospital-acquired infection in the US. During pneumonia and non-pneumonia severe illness, respiratory tract secretions become enriched with the serine protease neutrophil elastase (NE). Several NE activities promote onset and severity of NP. NE in the airways causes proteolytic tissue damage, augments inflammation, may promote invasion of respiratory epithelium by bacteria, and disrupts respiratory epithelial barrier function. These NE activities culminate in enhanced bacterial replication, impaired gas exchange, fluid intrusion into the airways, and loss of bacterial containment that can result in bacteremia. Therefore, neutralizing NE activity may reduce the frequency and severity of NP. We evaluated human alpha-1 antitrypsin (AAT), the prototype endogenous NE inhibitor, as a suppressor of bacterial pneumonia and pneumonia-related pathogenesis. In AAT+/+ transgenic mice that express human AAT in lungs, mortality due to Pseudomonas aeruginosa (P.aer) pneumonia was reduced 90% compared to non-transgenic control animals. Exogenous human AAT given to non-transgenic mice also significantly reduced P.aer pneumonia mortality. P.aer-infected AAT+/+ mice demonstrated reduced lung tissue damage, decreased bacterial concentrations in lungs and blood, and diminished circulating cytokine concentrations compared to infected non-transgenic mice. In vitro, AAT suppressed P.aer internalization into respiratory epithelial cells and inhibited NE or P.aer-induced disruption of epithelial cell barrier function. The beneficial effects of human AAT in murine P.aer pneumonia raise the possibility of AAT use as a prophylactic treatment for NP in humans, and suggest a role for AAT as an innate immune mediator.
PMCID: PMC3854847  PMID: 24350188
pneumonia; alpha-1 antitrypsin; cytokines; sepsis; Pseudomonas; neutrophil elastase
16.  Organization of the BcgI restriction–modification protein for the transfer of one methyl group to DNA 
Nucleic Acids Research  2012;41(1):405-417.
The Type IIB restriction–modification protein BcgI contains A and B subunits in a 2:1 ratio: A has the active sites for both endonuclease and methyltransferase functions while B recognizes the DNA. Like almost all Type IIB systems, BcgI needs two unmethylated sites for nuclease activity; it cuts both sites upstream and downstream of the recognition sequence, hydrolyzing eight phosphodiester bonds in a single synaptic complex. This complex may incorporate four A2B protomers to give the eight catalytic centres (one per A subunit) needed to cut all eight bonds. The BcgI recognition sequence contains one adenine in each strand that can be N6-methylated. Although most DNA methyltransferases operate at both unmethylated and hemi-methylated sites, BcgI methyltransferase is only effective at hemi-methylated sites, where the nuclease component is inactive. Unlike the nuclease, the methyltransferase acts at solitary sites, functioning catalytically rather than stoichiometrically. Though it transfers one methyl group at a time, presumably through a single A subunit, BcgI methyltransferase can be activated by adding extra A subunits, either individually or as part of A2B protomers, which indicates that it requires an assembly containing at least two A2B units.
PMCID: PMC3592466  PMID: 23147004
17.  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
18.  Determination of DNA sequences containing methylcytosine in Bacillus subtilis Marburg. 
Journal of Bacteriology  1985;163(2):573-579.
The methylcytosine-containing sequences in the DNA of Bacillus subtilis 168 Marburg (restriction-modification type BsuM) were determined by three different methods: (i) examination of in vivo-methylated DNA by restriction enzyme digestion and, whenever possible, analysis for methylcytosine at the 5' end; (ii) methylation in vitro of unmethylated DNA with B. subtilis DNA methyltransferase and determination of the methylated sites; and (iii) the methylatability of unmethylated DNA by B. subtilis methyltransferase after potential sites have been destroyed by digestion with restriction endonucleases. The results obtained by these methods, taken together, show that methylcytosine was present only within the sequence 5'-TCGA-3'. The presence of methylcytosine at the 5' end of the DNA fragments generated by restriction endonuclease AsuII digestion and the fact that in vivo-methylated DNA could not be digested by the enzyme XhoI showed that the recognition sequences of these two enzymes contained methylcytosine. As these two enzymes recognized a similar sequence containing a 5' pyrimidine (Py) and a 3' purine (Pu), 5'-PyTCGAPu-3', the possibility that methylcytosine is present in the complementary sequences 5'-TTCGAG-3' and 5'-CTCGAA-3' was postulated. This was verified by the methylation in vitro, with B. subtilis enzyme, of a 2.6-kilobase fragment of lambda DNA containing two such sites and devoid of AsuII or XhoI recognition sequences. By analyzing the methylatable sites, it was found that in one of the two PyTCGAPu sequences, cytosine was methylated in vitro in both DNA strands. It is concluded that the sequence 5'-PyTCGAPu-3' is methylated by the DNA methyltransferase (of cytosine) of B. subtilis Marburg.
PMCID: PMC219160  PMID: 2991196
19.  Methylation by a mutant T2 DNA [N6-adenine] methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage 
Nucleic Acids Research  2001;29(7):1484-1490.
Properties of a mutant bacteriophage T2 DNA [N6-adenine] methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher kcat in methylating canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes. In agreement with these steady-state kinetic data, when bacteriophage λ DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient. Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences. The feasibility of using the mutant enzyme in RARE cleavage with BclI and EcoRV endonucleases has been shown on phage λ DNA and with BclI and DpnII endonucleases on yeast chromosomal DNA embedded in agarose.
PMCID: PMC31273  PMID: 11266550
20.  Exposure to chronic mild stress differentially alters CRH and AVP mRNA expression in the stress-responsive neurocircuitry of male and female rats prenatally exposed to alcohol 
Prenatal alcohol exposure (PAE) results in dysregulation of the offspring hypothalamic-pituitary-adrenal (HPA) axis, increasing sensitivity to stressors and vulnerability to stress-related disorders. We have previously shown that exposure to chronic mild stress (CMS) in adulthood significantly increases anxiety-like behaviors (elevated plus maze) in PAE males and females compared to controls. To explore neurobiological mechanisms linking HPA dysregulation and altered anxiety-like behavior, we investigated neuropeptide [corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP)] expression in brain areas involved in the stress neurocircuitry of animals from this previous behavioral study.
Adult PAE, pair-fed (PF), and ad-libitum fed control (C) male and female offspring exposed to CMS or remaining undisturbed (Non-CMS) were terminated 30 min following behavioral testing.
In the paraventricular nucleus, CMS increased CRH mRNA levels in PAE compared to PF and C males, and increased AVP mRNA levels in PAE compared to C males, with no differential effects for CRH or AVP in females. In the central nucleus of the amygdala, there was an increase in CRH mRNA expression overall, regardless of CMS condition or sex, in PAE compared to C animals. Moreover, in PF males, CMS increased AVP mRNA levels in the paraventricular nucleus, resulting in a decreased CRH/AVP ratio compared to PAE males, and decreased amygdala CRH mRNA compared to that in the Non-CMS condition.
CMS differentially altered central HPA peptide expression in PAE and PF animals compared to their control counterparts, with a possible shift toward greater CRH mediation of HPA regulation in PAE males, and greater AVP mediation of HPA regulation in PF males. However, changes in CRH and AVP expression do not align fully with the anxiogenic profile observed in our previous behavior study, suggesting that other neuronal substrates and limbic forebrain regions also contribute to increased anxiety-like behavior following CMS.
PMCID: PMC4712115  PMID: 26578428
Prenatal Alcohol (Ethanol) Exposure; Hypothalamic-Pituitary-Adrenal (HPA) Axis; Corticotropin-Releasing Hormone (CRH); Arginine Vasopressin (AVP); Amygdala
21.  Sequence and substrate specificity of isolated DNA methylases from Escherichia coli C. 
Journal of Bacteriology  1983;153(1):274-280.
Two DNA methylase activities of Escherichia coli C, the mec (designates DNA-cytosine-methylase gene, which is also designated dcm) and dam gene products, were physically separated by DEAE-cellulose column chromatography. The sequence and substrate specificity of the two enzymes were studied in vitro. The experiments revealed that both enzymes show their expected sequence specificity under in vitro conditions, methylating symmetrically on both DNA strands. The mec enzyme methylates exclusively the internal cytosine residue of CCATGG sequences, and the dam enzyme methylates adenine residues at GATC sites. Substrate specificity experiments revealed that both enzymes methylate in vitro unmethylated duplex DNA as efficiently as hemimethylated DNA. The results of these experiments suggest that the methylation at a specific site takes place by two independent events. A methyl group in a site on one strand of the DNA does not facilitate the methylation of the same site on the opposite strand. With the dam methylase it was found that the enzyme is incapable of methylating GATC sites located at the ends of DNA molecules.
PMCID: PMC217366  PMID: 6336735
22.  Temperate Myxococcus xanthus phage Mx8 encodes a DNA adenine methylase, Mox. 
Journal of Bacteriology  1997;179(13):4254-4263.
Temperate bacteriophage Mx8 of Myxococcus xanthus encapsidates terminally repetitious DNA, packaged as circular permutations of its 49-kbp genome. During both lytic and lysogenic development, Mx8 expresses a nonessential DNA methylase, Mox, which modifies adenine residues in occurrences of XhoI and PstI recognition sites, CTCGAG and CTGCAG, respectively, on both phage DNA and the host chromosome. The mox gene is necessary for methylase activity in vivo, because an amber mutation in the mox gene abolishes activity. The mox gene is the only phage gene required for methylase activity in vivo, because ectopic expression of mox as part of the M. xanthus mglBA operon results in partial methylation of the host chromosome. The predicted amino acid sequence of Mox is related most closely to that of the methylase involved in the cell cycle control of Caulobacter crescentus. We speculate that Mox acts to protect Mx8 phage DNA against restriction upon infection of a subset of natural M. xanthus hosts. One natural isolate of M. xanthus, the lysogenic source of related phage Mx81, produces a restriction endonuclease with the cleavage specificity of endonuclease BstBI.
PMCID: PMC179247  PMID: 9209041
23.  Synthesis and properties of oligodeoxynucleotides containing the analogue 2'-deoxy-4'-thiothymidine. 
Nucleic Acids Research  1993;21(15):3485-3491.
The 2'-deoxythymidine analogue 2'-deoxy-4'-thiothymidine has been incorporated, using standard methodology, into a series of dodecadeoxynucleotides containing the EcoRV restriction endonuclease recognition site (GATATC). The stability of these oligodeoxynucleotides and their ability to act as substrates for the restriction endonuclease and associated methylase have been compared with a normal unmodified oligodeoxynucleotide. No problems were encountered in the synthesis despite the presence of a potentially oxidisable sulfur atom in the sugar ring. The analogue had very little effect on the melting temperature of the self-complementary oligoeoxynucleotides so synthesised and all had a CD spectrum compatible with a B-DNA structure. The oligodeoxynucleotide containing one analogue in each strand within the recognition site, adjacent to the bond to be cleaved (i.e. GAXATC, where X is 2'-deoxy-4'-thiothymidine), was neither a substrate for the endonuclease nor was recognized by the associated methylase. When still within the recognition hexanucleotide but two further residues removed from the site of cleavage (i.e. GATAXC), the oligodeoxynucleotide was a poor substrate for both the endonuclease and methylase. Binding of the oligodeoxynucleotide to the endonuclease was unaffected but the kcat value was only 0.03% of the value obtained for the parent oligodeoxynucleotide. These results show that the incorporation of 2'-deoxy-4'-thionucleosides into synthetic oligodeoxynucleotides may shed light on subtle interactions between proteins and their normal substrates and may also show why 2'-deoxy-4'-thiothymidine itself is so toxic in cell culture.
PMCID: PMC331449  PMID: 8346027
24.  Differential sensitivity to methylated DNA by ETS-family transcription factors is intrinsically encoded in their DNA-binding domains 
Nucleic Acids Research  2016;44(18):8671-8681.
Transactivation by the ETS family of transcription factors, whose members share structurally conserved DNA-binding domains, is variably sensitive to methylation of their target genes. The mechanism by which DNA methylation controls ETS proteins remains poorly understood. Uncertainly also pervades the effects of hemi-methylated DNA, which occurs following DNA replication and in response to hypomethylating agents, on site recognition by ETS proteins. To address these questions, we measured the affinities of two sequence-divergent ETS homologs, PU.1 and Ets-1, to DNA sites harboring a hemi- and fully methylated CpG dinucleotide. While the two proteins bound unmethylated DNA with indistinguishable affinity, their affinities to methylated DNA are markedly heterogeneous and exhibit major energetic coupling between the two CpG methylcytosines. Analysis of simulated DNA and existing co-crystal structures revealed that hemi-methylation induced non-local backbone and groove geometries that were not conserved in the fully methylated state. Indirect readout of these perturbations was differentially achieved by the two ETS homologs, with the distinctive interfacial hydration in PU.1/DNA binding moderating the inhibitory effects of DNA methylation on binding. This data established a biophysical basis for the pioneering properties associated with PU.1, which robustly bound fully methylated DNA, but not Ets-1, which was substantially inhibited.
PMCID: PMC5062964  PMID: 27270080
25.  Investigation of restriction-modification enzymes from M. varians RFL19 with a new type of specificity toward modification of substrate. 
Nucleic Acids Research  1985;13(16):5727-5746.
The characterization of MvaI restriction-modification enzymes, isolated from Micrococcus varians RFL19, is reported. Both enzymes recognize the 5'CC decreases (A/T)GG nucleotide sequence. The endonuclease cleaves the sequence at the position indicated by the arrow, whereas the methylase modifies the internal cytosine, yielding N4-methylcytosine. This type of modification protects the substrate from R.MvaI cleavage. 5-Methylcytosine in the same position of the recognition sequence does not protect the substrate from R.MvaI cleavage. R.MvaI proved to be the first example of a restriction endonuclease differentiating the position of the methyl group in the heterocyclic ring of cytosine, located in the same site of the recognition sequence. M.MvaI modifies DNA dcm+ in vitro yielding N4,5-dimethylcytosine. N4-methylcytosine cannot be differentiated from cytosine using the Maxam-Gilbert DNA sequencing procedure.
PMCID: PMC321908  PMID: 2994011

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