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Norbert O Reich - One of the best experts on this subject based on the ideXlab platform.
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the cell cycle regulated DNA Adenine Methyltransferase ccrm opens a bubble at its DNA recognition site
Nature Communications, 2019Co-Authors: John R Horton, Xing Zhang, Norbert O Reich, Clayton B Woodcock, Sifa B Opot, Xiaodong ChengAbstract:The Caulobacter crescentus cell cycle-regulated DNA Methyltransferase (CcrM) methylates the Adenine of hemimethylated GANTC after replication. Here we present the structure of CcrM in complex with double-stranded DNA containing the recognition sequence. CcrM contains an N-terminal Methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the Methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the 5-base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson–Crick polar hydrogen bonds to ensure high enzymatic specificity. CcrM is a cell cycle-regulated DNA Methyltransferase that methylates an Adenine within a specific sequence following replication in the gram negative bacterium Caulobacter crescentus. Here the authors present a crystal structure of DNA-bound CcrM that reveals the molecular mechanism leading to sequence-specific methylation.
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Integrated rate laws for processive and distributive enzymatic turnover
Journal of Chemical Physics, 2019Co-Authors: Itay Barel, Norbert O Reich, Frank L H BrownAbstract:Recently derived steady-state differential rate laws for the catalytic turnover of molecules containing two substrate sites are reformulated as integrated rate laws. The analysis applies to a broad class of Markovian dynamic models, motivated by the varied and often complex mechanisms associated with DNA modifying enzymes. Analysis of experimental data for the methylation kinetics of DNA by Dam (DNA Adenine Methyltransferase) is drastically improved through the use of integrated rate laws. Data that are too noisy for fitting to differential predictions are reliably interpreted through the integrated rate laws.Recently derived steady-state differential rate laws for the catalytic turnover of molecules containing two substrate sites are reformulated as integrated rate laws. The analysis applies to a broad class of Markovian dynamic models, motivated by the varied and often complex mechanisms associated with DNA modifying enzymes. Analysis of experimental data for the methylation kinetics of DNA by Dam (DNA Adenine Methyltransferase) is drastically improved through the use of integrated rate laws. Data that are too noisy for fitting to differential predictions are reliably interpreted through the integrated rate laws.
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specificity versus processivity in the sequential modification of DNA a study of DNA Adenine Methyltransferase
Journal of Physical Chemistry B, 2018Co-Authors: Itay Barel, Norbert O Reich, Brigitte Naughton, Frank L H BrownAbstract:A detailed analysis is carried out on both published experimental results and new experiments for the methylation kinetics of two-site DNA substrates (with site separations between 100 and 800 bp) catalyzed by bacterial DNA Adenine Methyltransferase (Dam). A previously reported rate enhancement for the second methylation event (relative to that of the first methylation) is shown to result from elevated substrate specificity for singly methylated DNA over that of unmethylated DNA and not processive turnover of both sites by the same copy of Dam. An elementary model is suggested that cleanly fits the experimental data over a broad range of intersite separations. The model hypothesizes a looping mediated interference between competing unmethylated Dam sites on the same DNA strand.
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Mechanisms of Protein Translocation on DNA Are Differentially Responsive to Water Activity
Biochemistry, 2016Co-Authors: Brigitte Naughton, Norbert O ReichAbstract:Water plays important but poorly understood roles in the functions of most biomolecules. We are interested in understanding how proteins use diverse search mechanisms to locate specific sites on DNA; here we present a study of the role of closely associated waters in diverse translocation mechanisms. The bacterial DNA Adenine Methyltransferase, Dam, moves across large segments of DNA using an intersegmental hopping mechanism, relying in part on movement through bulk water. In contrast, other proteins, such as the bacterial restriction endonuclease EcoRI, rely on a sliding mechanism, requiring the protein to stay closely associated with DNA. Here we probed how these two mechanistically distinct proteins respond to well-characterized osmolytes, dimethyl sulfoxide (DMSO), and glycerol. The ability of Dam to move over large segments of DNA is not impacted by either osmolyte, consistent with its minimal reliance on a sliding mechanism. In contrast, EcoRI endonuclease translocation is significantly enhanced by ...
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DNA Adenine Methyltransferase facilitated diffusion is enhanced by protein DNA roadblock complexes that induce DNA looping
Biochemistry, 2015Co-Authors: Adam J Pollak, Norbert O ReichAbstract:The genomes of all cells are intimately associated with proteins, which are important for compaction, scaffolding, and gene regulation. Here we show that pre-existing protein–DNA complexes (roadblocks) diminish and―interestingly―enhance the ability of particular sequence-specific proteins to move along DNA to locate their binding sites. We challenge the bacterial DNA Adenine Methyltransferase (Dam, recognizes 5′-GATC-3′) with tightly bound EcoRV ENase–DNA complexes, which bend DNA. A single EcoRV roadblock does not alter processive (multiple modifications) methylation by Dam. This result disfavors a reliance on heavily touted mechanisms involving sliding or short hops for Dam. Specific conformations of two EcoRV roadblocks cause an increase in processivity. The histone-like leucine-responsive regulatory protein (Lrp) binds DNA nonspecifically as an octamer, and also increases Dam’s processivity. These results can be explained by our prior demonstration that Dam moves over large regions (>300 bp) within a ...
Xiaodong Cheng - One of the best experts on this subject based on the ideXlab platform.
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the cell cycle regulated DNA Adenine Methyltransferase ccrm opens a bubble at its DNA recognition site
Nature Communications, 2019Co-Authors: John R Horton, Xing Zhang, Norbert O Reich, Clayton B Woodcock, Sifa B Opot, Xiaodong ChengAbstract:The Caulobacter crescentus cell cycle-regulated DNA Methyltransferase (CcrM) methylates the Adenine of hemimethylated GANTC after replication. Here we present the structure of CcrM in complex with double-stranded DNA containing the recognition sequence. CcrM contains an N-terminal Methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the Methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the 5-base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson–Crick polar hydrogen bonds to ensure high enzymatic specificity. CcrM is a cell cycle-regulated DNA Methyltransferase that methylates an Adenine within a specific sequence following replication in the gram negative bacterium Caulobacter crescentus. Here the authors present a crystal structure of DNA-bound CcrM that reveals the molecular mechanism leading to sequence-specific methylation.
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structures of escherichia coli DNA Adenine Methyltransferase dam in complex with a non gatc sequence potential implications for methylation independent transcriptional repression
Nucleic Acids Research, 2015Co-Authors: John R Horton, Xing Zhang, Robert Blumenthal, Xiaodong ChengAbstract:DNA Adenine Methyltransferase (Dam) is widespread and conserved among the γ-proteobacteria. Methylation of the Ade in GATC sequences regulates diverse bacterial cell functions, including gene expression, mismatch repair and chromosome replication. Dam also controls virulence in many pathogenic Gram-negative bacteria. An unexplained and perplexing observation about Escherichia coli Dam (EcoDam) is that there is no obvious relationship between the genes that are transcriptionally responsive to Dam and the promoter-proximal presence of GATC sequences. Here, we demonstrate that EcoDam interacts with a 5-base pair non-cognate sequence distinct from GATC. The crystal structure of a non-cognate complex allowed us to identify a DNA binding element, GTYTA/TARAC (where Y = C/T and R = A/G). This element immediately flanks GATC sites in some Dam-regulated promoters, including the Pap operon which specifies pyelonephritis-associated pili. In addition, Dam interacts with near-cognate GATC sequences (i.e. 3/4-site ATC and GAT). Taken together, these results imply that Dam, in addition to being responsible for GATC methylation, could also function as a methylation-independent transcriptional repressor.
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two alternative conformations of s adenosyl l homocysteine bound to escherichia coli DNA Adenine Methyltransferase and the implication of conformational changes in regulating the catalytic cycle
Journal of Biological Chemistry, 2007Co-Authors: Kirsten Liebert, John R Horton, Xiaodong Cheng, Sanjay Chahar, Marcella Orwick, Albert JeltschAbstract:Abstract The crystal structure of the Escherichia coli DNA Adenine Methyltransferase (EcoDam) in a binary complex with the cofactor product S-adenosyl-l-homocysteine (AdoHcy) unexpectedly showed the bound AdoHcy in two alternative conformations, extended or folded. The extended conformation represents the catalytically competent conformation, identical to that of EcoDam-DNA-AdoHcy ternary complex. The folded conformation prevents catalysis, because the homocysteine moiety occupies the target Ade binding pocket. The largest difference between the binary and ternary structures is in the conformation of the N-terminal hexapeptide (9KWAGGK14). Cofactor binding leads to a strong change in the fluorescence of Trp10, whose indole ring approaches the cofactor by 3.3A. Stopped-flow kinetics and AdoMet cross-linking studies indicate that the cofactor prefers binding to the enzyme after preincubation with DNA. In the presence of DNA, AdoMet binding is ∼2-fold stronger than AdoHcy binding. In the binary complex the side chain of Lys14 is disordered, whereas Lys14 stabilizes the active site in the ternary complex. Fluorescence stopped-flow experiments indicate that Lys14 is important for EcoDam binding of the extrahelical target base into the active site pocket. This suggests that the hexapeptide couples specific DNA binding (Lys9), AdoMet binding (Trp10), and insertion of the flipped target base into the active site pocket (Lys14).
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structure and substrate recognition of the escherichia coli DNA Adenine Methyltransferase
Journal of Molecular Biology, 2006Co-Authors: John R Horton, Kirsten Liebert, Albert Jeltsch, Miklos Bekes, Xiaodong ChengAbstract:The structure of the Escherichia coli Dam DNA-(Adenine-N6)-Methyltransferase in complex with cognate DNA was determined at 1.89 A resolution in the presence of S -adenosyl- l -homocysteine. DNA recognition and the dynamics of base-flipping were studied by site-directed mutagenesis, DNA methylation kinetics and fluorescence stopped-flow experiments. Our data illustrate the mechanism of coupling of DNA recognition and base-flipping. Contacts to the non-target strand in the second (3′) half of the GATC site are established by R124 to the fourth base-pair, and by L122 and P134 to the third base-pair. The aromatic ring of Y119 intercalates into the DNA between the second and third base-pairs, which is essential for base-flipping to occur. Compared to previous published structures of bacteriophage T4 Dam, three major new observations are made in E. coli Dam. (1) The first Gua is recognized by K9, removal of which abrogates the first base-pair recognition. (2) The flipped target Ade binds to the surface of EcoDam in the absence of S -adenosyl- l -methionine, which illustrates a possible intermediate in the base-flipping pathway. (3) The orphaned Thy residue displays structural flexibility by adopting an extrahelical or intrahelical position where it is in contact to N120.
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Transition from Nonspecific to Specific DNA Interactions along the Substrate-Recognition Pathway of Dam Methyltransferase
Cell, 2005Co-Authors: John R Horton, Stanley Hattman, Kirsten Liebert, Albert Jeltsch, Xiaodong ChengAbstract:DNA Methyltransferases methylate target bases within specific nucleotide sequences. Three structures are described for bacteriophage T4 DNA-Adenine Methyltransferase (T4Dam) in ternary complexes with partially and fully specific DNA and a methyl-donor analog. We also report the effects of substitutions in the related Escherichia coli DNA Methyltransferase (EcoDam), altering residues corresponding to those involved in specific interaction with the canonical GATC target sequence in T4Dam. We have identified two types of protein-DNA interactions: discriminatory contacts, which stabilize the transition state and accelerate methylation of the cognate site, and anti-discriminatory contacts, which do not significantly affect methylation of the cognate site but disfavor activity at noncognate sites. These structures illustrate the transition in enzyme-DNA interaction from nonspecific to specific interaction, suggesting that there is a temporal order for formation of specific contacts.
John R Horton - One of the best experts on this subject based on the ideXlab platform.
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the cell cycle regulated DNA Adenine Methyltransferase ccrm opens a bubble at its DNA recognition site
Nature Communications, 2019Co-Authors: John R Horton, Xing Zhang, Norbert O Reich, Clayton B Woodcock, Sifa B Opot, Xiaodong ChengAbstract:The Caulobacter crescentus cell cycle-regulated DNA Methyltransferase (CcrM) methylates the Adenine of hemimethylated GANTC after replication. Here we present the structure of CcrM in complex with double-stranded DNA containing the recognition sequence. CcrM contains an N-terminal Methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the Methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the 5-base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson–Crick polar hydrogen bonds to ensure high enzymatic specificity. CcrM is a cell cycle-regulated DNA Methyltransferase that methylates an Adenine within a specific sequence following replication in the gram negative bacterium Caulobacter crescentus. Here the authors present a crystal structure of DNA-bound CcrM that reveals the molecular mechanism leading to sequence-specific methylation.
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structures of escherichia coli DNA Adenine Methyltransferase dam in complex with a non gatc sequence potential implications for methylation independent transcriptional repression
Nucleic Acids Research, 2015Co-Authors: John R Horton, Xing Zhang, Robert Blumenthal, Xiaodong ChengAbstract:DNA Adenine Methyltransferase (Dam) is widespread and conserved among the γ-proteobacteria. Methylation of the Ade in GATC sequences regulates diverse bacterial cell functions, including gene expression, mismatch repair and chromosome replication. Dam also controls virulence in many pathogenic Gram-negative bacteria. An unexplained and perplexing observation about Escherichia coli Dam (EcoDam) is that there is no obvious relationship between the genes that are transcriptionally responsive to Dam and the promoter-proximal presence of GATC sequences. Here, we demonstrate that EcoDam interacts with a 5-base pair non-cognate sequence distinct from GATC. The crystal structure of a non-cognate complex allowed us to identify a DNA binding element, GTYTA/TARAC (where Y = C/T and R = A/G). This element immediately flanks GATC sites in some Dam-regulated promoters, including the Pap operon which specifies pyelonephritis-associated pili. In addition, Dam interacts with near-cognate GATC sequences (i.e. 3/4-site ATC and GAT). Taken together, these results imply that Dam, in addition to being responsible for GATC methylation, could also function as a methylation-independent transcriptional repressor.
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two alternative conformations of s adenosyl l homocysteine bound to escherichia coli DNA Adenine Methyltransferase and the implication of conformational changes in regulating the catalytic cycle
Journal of Biological Chemistry, 2007Co-Authors: Kirsten Liebert, John R Horton, Xiaodong Cheng, Sanjay Chahar, Marcella Orwick, Albert JeltschAbstract:Abstract The crystal structure of the Escherichia coli DNA Adenine Methyltransferase (EcoDam) in a binary complex with the cofactor product S-adenosyl-l-homocysteine (AdoHcy) unexpectedly showed the bound AdoHcy in two alternative conformations, extended or folded. The extended conformation represents the catalytically competent conformation, identical to that of EcoDam-DNA-AdoHcy ternary complex. The folded conformation prevents catalysis, because the homocysteine moiety occupies the target Ade binding pocket. The largest difference between the binary and ternary structures is in the conformation of the N-terminal hexapeptide (9KWAGGK14). Cofactor binding leads to a strong change in the fluorescence of Trp10, whose indole ring approaches the cofactor by 3.3A. Stopped-flow kinetics and AdoMet cross-linking studies indicate that the cofactor prefers binding to the enzyme after preincubation with DNA. In the presence of DNA, AdoMet binding is ∼2-fold stronger than AdoHcy binding. In the binary complex the side chain of Lys14 is disordered, whereas Lys14 stabilizes the active site in the ternary complex. Fluorescence stopped-flow experiments indicate that Lys14 is important for EcoDam binding of the extrahelical target base into the active site pocket. This suggests that the hexapeptide couples specific DNA binding (Lys9), AdoMet binding (Trp10), and insertion of the flipped target base into the active site pocket (Lys14).
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structure and substrate recognition of the escherichia coli DNA Adenine Methyltransferase
Journal of Molecular Biology, 2006Co-Authors: John R Horton, Kirsten Liebert, Albert Jeltsch, Miklos Bekes, Xiaodong ChengAbstract:The structure of the Escherichia coli Dam DNA-(Adenine-N6)-Methyltransferase in complex with cognate DNA was determined at 1.89 A resolution in the presence of S -adenosyl- l -homocysteine. DNA recognition and the dynamics of base-flipping were studied by site-directed mutagenesis, DNA methylation kinetics and fluorescence stopped-flow experiments. Our data illustrate the mechanism of coupling of DNA recognition and base-flipping. Contacts to the non-target strand in the second (3′) half of the GATC site are established by R124 to the fourth base-pair, and by L122 and P134 to the third base-pair. The aromatic ring of Y119 intercalates into the DNA between the second and third base-pairs, which is essential for base-flipping to occur. Compared to previous published structures of bacteriophage T4 Dam, three major new observations are made in E. coli Dam. (1) The first Gua is recognized by K9, removal of which abrogates the first base-pair recognition. (2) The flipped target Ade binds to the surface of EcoDam in the absence of S -adenosyl- l -methionine, which illustrates a possible intermediate in the base-flipping pathway. (3) The orphaned Thy residue displays structural flexibility by adopting an extrahelical or intrahelical position where it is in contact to N120.
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Transition from Nonspecific to Specific DNA Interactions along the Substrate-Recognition Pathway of Dam Methyltransferase
Cell, 2005Co-Authors: John R Horton, Stanley Hattman, Kirsten Liebert, Albert Jeltsch, Xiaodong ChengAbstract:DNA Methyltransferases methylate target bases within specific nucleotide sequences. Three structures are described for bacteriophage T4 DNA-Adenine Methyltransferase (T4Dam) in ternary complexes with partially and fully specific DNA and a methyl-donor analog. We also report the effects of substitutions in the related Escherichia coli DNA Methyltransferase (EcoDam), altering residues corresponding to those involved in specific interaction with the canonical GATC target sequence in T4Dam. We have identified two types of protein-DNA interactions: discriminatory contacts, which stabilize the transition state and accelerate methylation of the cognate site, and anti-discriminatory contacts, which do not significantly affect methylation of the cognate site but disfavor activity at noncognate sites. These structures illustrate the transition in enzyme-DNA interaction from nonspecific to specific interaction, suggesting that there is a temporal order for formation of specific contacts.
Peter L Roach - One of the best experts on this subject based on the ideXlab platform.
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inhibition of yersinia pestis DNA Adenine Methyltransferase in vitro by a stibonic acid compound identification of a potential novel class of antimicrobial agents
British Journal of Pharmacology, 2013Co-Authors: Jennifer C Mckelvie, Peter L Roach, Jenny E Harmer, M I Richards, T S Milne, Pcf OystonAbstract:Background and Purpose Multiple antibiotic resistant strains of plague are emerging, driving a need for the development of novel antibiotics effective against Yersinia pestis. DNA Adenine methylation regulates numerous fundamental processes in bacteria and alteration of DNA Adenine methlytransferase (Dam) expression is attenuating for several pathogens, including Y. pestis. The lack of a functionally similar enzyme in humans makes Dam a suitable target for development of novel therapeutics for plague. Experimental Approach Compounds were evaluated for their ability to inhibit Dam activity in a high-throughput screening assay. DNA was isolated from Yersinia grown in the presence of lead compounds and restricted to determine the effect of inhibitors on DNA methylation. Transcriptional analysis was undertaken to determine the effect of an active inhibitor on virulence-associated phenotypes. Key Results We have identified a series of aryl stibonic acids which inhibit Dam in vitro. The most active, 4-stibonobenzenesulfonic acid, exhibited a competitive mode of inhibition with respect to DNA and a Ki of 6.46 nM. One compound was found to inhibit DNA methylation in cultured Y. pestis. The effects of this inhibition on the physiology of the cell were widespread, and included altered expression of known virulence traits, including iron acquisition and Type III secretion. Conclusions and Implications We have identified a novel class of potent Dam inhibitors. Treatment of bacterial cell cultures with these inhibitors resulted in a decrease in DNA methylation. Expression of virulence factors was affected, suggesting these inhibitors may attenuate bacterial infectivity and function as antibiotics.
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direct and continuous fluorescence based measurements of pyrococcus horikoshii DNA n 6 Adenine Methyltransferase activity
Analytical Biochemistry, 2011Co-Authors: Michael D Maynardsmith, R J K Wood, Jennifer C Mckelvie, Jenny E Harmer, Rohan T Ranasinghe, Claire L Williams, Danielle M Coomber, Alexandra F Stares, Peter L RoachAbstract:Abstract N-6 methylation of Adenine destabilises duplex DNA and this can increase the proportion of DNA that dissociates into single strands. We have investigated utilising this property to measure the DNA Adenine Methyltransferase-catalyzed conversion of hemimethylated to fully methylated DNA through a simple, direct, fluorescence-based assay. The effects of methylation on the kinetics and thermodynamics of hybridisation were measured by comparing a fully methylated oligonucleotide product and a hemimethylated oligonucleotide substrate using a 13-bp duplex labeled on adjacent strands with a fluorophore (fluorescein) and quencher (dabcyl). Enzymatic methylation of the hemimethylated GATC site resulted in destabilisation of the duplex, increasing the proportion of dissociated DNA, and producing an observable increase in fluorescence. The assay provides a direct measurement of methylation rate in real time and is highly reproducible, with a coefficient of variance over 48 independent measurements of 3.6%. DNA methylation rates can be measured as low as 3.55 ± 1.84 fmol s−1 in a 96-well plate format, and the assay has been used to kinetically characterise the Pyrococcus horikoshii DNA Adenine Methyltransferase.
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kinetic analysis of yersinia pestis DNA Adenine Methyltransferase activity using a hemimethylated molecular break light oligonucleotide
PLOS ONE, 2007Co-Authors: R J K Wood, Michael D Maynardsmith, Victoria L Robinson, Petra C F Oyston, Richard W Titball, Peter L RoachAbstract: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 0.7160.07 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
Martin Marinus - One of the best experts on this subject based on the ideXlab platform.
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DNA methylation and mutator genes in Escherichia coli K-12.
Mutation Research-reviews in Mutation Research, 2010Co-Authors: Martin MarinusAbstract:Mutator strains of Escherichia coli have been used to define mechanisms that account for the high fidelity of chromosome duplication and chromosome stability. Mutant strains defective in post-replicative mismatch repair display a strong mutator phenotype consistent with a role for correction of mismatches arising from replication errors. Inactivation of the gene (dam) encoding DNA Adenine Methyltransferase results in a mutator phenotype consistent with a role for DNA methylation in strand discrimination during mismatch repair. This review gives a personal perspective on the discovery of dam mutants in E. coli and their relationship to mismatch repair and mutator phenotypes.
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YhdJ, a Nonessential CcrM-Like DNA Methyltransferase of Escherichia coli and Salmonella enterica
Journal of Bacteriology, 2007Co-Authors: Sarah E. Broadbent, Martin Marinus, Roberto Balbontín, Josep Casadesús, Marjan W. Van Der WoudeAbstract:The Caulobacter crescentus DNA Adenine Methyltransferase CcrM and its homologs in the α-Proteobacteria are essential for viability. CcrM is 34% identical to the yhdJ gene products of Escherichia coli and Salmonella enterica. This study provides evidence that the E. coli yhdJ gene encodes a DNA Adenine Methyltransferase. In contrast to an earlier report, however, we show that yhdJ is not an essential gene in either E. coli or S. enterica.
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analysis of global gene expression and double strand break formation in DNA Adenine Methyltransferase and mismatch repair deficient escherichia coli
Journal of Bacteriology, 2005Co-Authors: Jennifer L Robbinsmanke, Zoran Z Zdraveski, Martin Marinus, John M EssigmannAbstract:DNA Adenine methylation by DNA Adenine Methyltransferase (Dam) in Escherichia coli plays an important role in processes such as DNA replication initiation, gene expression regulation, and mismatch repair. In addition, E. coli strains deficient in Dam are hypersensitive to DNA-damaging agents. We used genome microarrays to compare the transcriptional profiles of E. coli strains deficient in Dam and mismatch repair (dam, dam mutS, and mutS mutants). Our results show that >200 genes are expressed at a higher level in the dam strain, while an additional mutation in mutS suppresses the induction of many of the same genes. We also show by microarray and semiquantitative real-time reverse transcription-PCR that both dam and dam mutS strains show derepression of LexA-regulated SOS genes as well as the up-regulation of other non-SOS genes involved in DNA repair. To correlate the level of SOS induction and the up-regulation of genes involved in recombinational repair with the level of DNA damage, we used neutral single-cell electrophoresis to determine the number of double-strand breaks per cell in each of the strains. We find that dam mutant E. coli strains have a significantly higher level of double-strand breaks than the other strains. We also observe a broad range in the number of double-strand breaks in dam mutant cells, with a minority of cells showing as many as 10 or more double-strand breaks. We propose that the up-regulation of recombinational repair in dam mutants allows for the efficient repair of double-strand breaks whose formation is dependent on functional mismatch repair.
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Dam methylation: coordinating cellular processes.
Current Opinion in Microbiology, 2005Co-Authors: Anders Løbner-olesen, Ole Skovgaard, Martin MarinusAbstract:GATC sequences in Escherichia coli DNA are methylated at the Adenine residue by DNA Adenine Methyltransferase (DamMT). These methylated residues and/or the level of DamMT can influence cellular functions such as gene transcription, DNA mismatch repair, initiation of chromosome replication and nucleoid structure. In certain bacteria, unlike E. coli, DamMT is essential for viability perhaps owing to its role in chromosome replication. DamMT has also been implicated as a virulence factor in bacterial pathogenesis. The origin and phylogeny of DamMT, based on sequenced genomes, has been deduced.
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recombination is essential for viability of an escherichia coli dam DNA Adenine Methyltransferase mutant
Journal of Bacteriology, 2000Co-Authors: Martin MarinusAbstract:expected. The inviability of a dam lexA (Ind 2 ) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB. This result indicates that of more than 20 SOS genes, only recA and ruvAB need to be derepressed to allow for dam mutant survival. The presence of mutS or mutL mutations allowed the construction of dam lexA (Ind 2 ) derivatives. The requirement for recA, recB, recC, ruvA, ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability of dam bacteria probably to repair DNA double-strand breaks. The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents. The dam gene of Escherichia coli encodes a DNA Methyltransferase that methylates Adenine in -GATC- sequences in double-stranded DNA (17). Mutant strains lacking this enzyme display a pleiotropic phenotype including increased mutability, hyperrecombination, and increased sensitivity to DNA-damaging agents. In addition, dam bacteria have an increased number of single-strand breaks in DNA compared to wild type. The phenotypes displayed by dam mutants are consistent with multiple roles of unmethylated, methylated, and hemimethylated -GATC- sequences in cellular physiology. These include regulation of gene expression and strand discrimination during replication-associated DNA mismatch repair (17). An additional feature of dam strains is inviability when combined with mutant alleles of recA, recB, recC, or noninducible (Ind 2 ) lexA (19). The lexA inviability suggests a requirement for derepression of one or more SOS genes. The SOS response is induced following treatments that damage DNA or inhibit DNA replication (6). About 20 genes (including recA, lexA, and ruvAB) that are negatively regulated by LexA are derepressed following cleavage of the LexA repressor. Treatments that induce the SOS regulon do so by activating the coprotease activity of RecA (“activated RecA”), resulting in LexA cleavage. RecA protein also catalyzes 39-single-strand invasion of homologous DNA and is, therefore, essential in the recombination process (15). Peterson et al. (24) showed that dam bacteria with a temperature-sensitive lexA allele were viable at 42°C but not at 30°C, indicating the requirement for derepressed expression of one or more LexA-regulated SOS genes. In addition, Peterson et al. (24) found higher basal-level expression (two- to sixfold) of several SOS genes (including recA, lexA, sulA, uvrA, uvrB, uvrD, dinD, and recF )i ndam mutants than in wild type. However, since other genes are also induced by LexA cleavage, it was not possible to determine which are required for dam viability. In the present communication, the SOS genes required for viability of dam strains have been identified. They are recA and ruvAB, the latter encoding enzymes that translocate Holliday junctions (15, 29). Two other non-SOS genes have also been identified. The recG gene product can also catalyze translocation (15), and dam recG mutants are probably inviable. It is also shown that expression of the ruvC gene product, a Holliday junction resolvase (15, 29), is also required for dam mutant viability. The requirement for recA, recB, recC, recG, ruvA, ruvB, and ruvC gene expression indicates that recombination is essential for dam mutant viability.