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1.
Nat Commun ; 10(1): 4600, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601797

RESUMO

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.


Assuntos
Caulobacter crescentus/enzimologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética
2.
J Chem Phys ; 150(24): 244120, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31255081

RESUMO

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.


Assuntos
DNA/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Metilação de DNA , Cinética , Cadeias de Markov , Modelos Químicos
3.
Nat Struct Mol Biol ; 26(5): 361-371, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31061526

RESUMO

Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a member of the seven-ß-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127-TRMT112, hereafter termed 'lysine methyltransferase 9' (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor-dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.


Assuntos
Proliferação de Células/fisiologia , Histonas/metabolismo , Lisina/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Linhagem Celular Tumoral , Dimerização , Histonas/química , Humanos , Masculino , Metilação , Metiltransferases/química , Metiltransferases/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/fisiologia
4.
ACS Appl Mater Interfaces ; 11(18): 16958-16964, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-30993969

RESUMO

Photoelectrochemical (PEC) sensing techniques have attracted considerable concerns because of the intrinsic merit of complete separation between the excitation light and responsive current but still remain a great challenge for further potential application. It is assigned to the scarcity of photoactive materials with narrow band gap, good biosafety, and high photon-to-electron conversion efficiency and unfavorable processing methods for photoactive materials on indium tin oxide. Herein, we employed a perylene-based polymer (PTC-NH2) with exceptional photoelectrical properties to develop a red-light-driven PEC sensor for ultrasensitive biosensing based on its superior electrostatic intercalation efficiency in double-stranded DNA to that in single-stranded DNA, with DNA adenine methyltransferase (Dam MTase) as the model target. The prepared PTC-NH2 was characterized by Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, and PEC techniques, and the results demonstrated that PTC-NH2 rather than metal oxides/metal sulfides/C3N4/metal complexes enjoyed the prominent capacity of converting light to current. Benefiting from the unique PEC properties of PTC-NH2 and target-initiated hybridization chain reaction (HCR) signal amplification, ultrasensitive detection of Dam MTase was accessibly realized with the detection limit of 0.015 U/mL, which is lower than that of PEC, electrochemical, or fluorescent biosensors previously reported. Furthermore, the proposed PEC sensor has been also applied in screening Dam MTase activity inhibitors. Therefore, the perylene-based PEC sensor exhibits great potential in early accurate diagnosis of DNA methylation-related diseases.


Assuntos
Técnicas Biossensoriais , DNA/química , Substâncias Intercalantes/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/isolamento & purificação , DNA de Cadeia Simples/efeitos da radiação , Técnicas Eletroquímicas/métodos , Humanos , Luz , Perileno/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Compostos de Estanho/química
5.
J Biol Chem ; 293(49): 19038-19046, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30323065

RESUMO

Two DNA methyltransferases, Dam and ß-class cell cycle-regulated DNA methyltransferase (CcrM), are key mediators of bacterial epigenetics. CcrM from the bacterium Caulobacter crescentus (CcrM C. crescentus, methylates adenine at 5'-GANTC-3') displays 105-107-fold sequence discrimination against noncognate sequences. However, the underlying recognition mechanism is unclear. Here, CcrM C. crescentus activity was either improved or mildly attenuated with substrates having one to three mismatched bp within or adjacent to the recognition site, but only if the strand undergoing methylation is left unchanged. By comparison, single-mismatched substrates resulted in up to 106-fold losses of activity with α (Dam) and γ-class (M.HhaI) DNA methyltransferases. We found that CcrM C. crescentus has a greatly expanded DNA-interaction surface, covering six nucleotides on the 5' side and eight nucleotides on the 3' side of its recognition site. Such a large interface may contribute to the enzyme's high sequence fidelity. CcrM C. crescentus displayed the same sequence discrimination with single-stranded substrates, and a surprisingly large (>107-fold) discrimination against ssRNA was largely due to the presence of two or more riboses within the cognate (DNA) site but not outside the site. Results from C-terminal truncations and point mutants supported our hypothesis that the recently identified C-terminal, 80-residue segment is essential for dsDNA recognition but is not required for single-stranded substrates. CcrM orthologs from Agrobacterium tumefaciens and Brucella abortus share some of these newly discovered features of the C. crescentus enzyme, suggesting that the recognition mechanism is conserved. In summary, CcrM C. crescentus uses a previously unknown DNA recognition mechanism.


Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/enzimologia , DNA Bacteriano/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Agrobacterium tumefaciens/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Pareamento Incorreto de Bases , Brucella abortus/enzimologia , Domínio Catalítico , Metilação de DNA , DNA Bacteriano/genética , Domínios Proteicos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química
6.
J Phys Chem B ; 122(39): 9061-9075, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30117741

RESUMO

The role of Mg2+ ions during precursor formation in DNA hydrolysis by the homodimeric restriction enzyme EcoRV was elucidated based on the 3D-reference interaction site model (RISM) theory and the molecular dynamics (MD) simulation. From an analysis of the spatial distribution of Mg2+ in an active site using 3D-RISM, we identified a new position for Mg2+ in the X-ray EcoRV-DNA complex structure ( 1rvb ). We refer to the position as site IV†. Site IV† is almost at the same position as that of a Ca2+ ion in the superimposed X-ray crystallographic active-site structure of the PvuII-DNA complex ( 1f0o ). 3D-RISM was also used to locate the position of water molecules, including the water nucleophile at the active site. MD simulations were carried out with the initial structure having two Mg2+ ions at site IV† and at site I*, experimentally identified by Horton et al., to find a stable complex structure in which the DNA fragment was rearranged to orient the scissile bond direction toward the water nucleophile. The equilibrium active-site structure of the EcoRV-DNA complex obtained from the MD simulation was similar to the superimposed X-ray crystallographic structure of the BamHI-DNA complex ( 2bam ). In the active-site structure, two metal ions have almost the same position (≤1.0 Å) as that of 2bam , and the scissile phosphate is twisted to orient the scissile bond toward the water nucleophile, as is the case in 2bam . We propose the equilibrium active-site structure obtained in this study as a precursor for the hydrolysis reaction of EcoRV.


Assuntos
DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Magnésio/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Água/metabolismo , Catálise , Domínio Catalítico , DNA/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Hidrólise , Magnésio/química , Simulação de Dinâmica Molecular , Ligação Proteica , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Água/química
7.
Biomed Res Int ; 2018: 5657085, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29789800

RESUMO

Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. Lactococcus raffinolactis BGTRK10-1 expresses LraI Type II restriction-modification enzyme, whose activity is similar to that shown for EcoRI; LraI methyltransferase protects DNA from EcoRI cleavage. The gene encoding LraI endonuclease was cloned and overexpressed in E. coli. Purified enzyme showed the highest specific activity at lower temperatures (between 13°C and 37°C) and was stable after storage at -20°C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity of LraI restriction enzyme was 100 mM or higher. The recognition and cleavage sequence for LraI restriction enzyme was determined as 5'-G/AATTC-3', indicating that LraI restriction enzyme is an isoschizomer of EcoRI. In the reaction buffer with a lower salt concentration, LraI exhibits star activity and specifically recognizes and cuts another alternative sequence 5'-A/AATTC-3', leaving the same sticky ends on fragments as EcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin of LraI restriction-modification system with previously described EcoRI-like restriction-modification systems.


Assuntos
Proteínas de Bactérias/metabolismo , Lactococcus/enzimologia , Lactococcus/genética , Proteínas Recombinantes/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração Osmolar , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética
8.
J Phys Chem B ; 122(3): 1112-1120, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29258307

RESUMO

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.


Assuntos
DNA/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , DNA/química , Metilação de DNA , Cinética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Especificidade por Substrato
9.
Nat Commun ; 8(1): 368, 2017 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-28848232

RESUMO

Quantitative analysis of the sequence determinants of transcription and translation regulation is relevant for systems and synthetic biology. To identify these determinants, researchers have developed different methods of screening random libraries using fluorescent reporters or antibiotic resistance genes. Here, we have implemented a generic approach called ELM-seq (expression level monitoring by DNA methylation) that overcomes the technical limitations of such classic reporters. ELM-seq uses DamID (Escherichia coli DNA adenine methylase as a reporter coupled with methylation-sensitive restriction enzyme digestion and high-throughput sequencing) to enable in vivo quantitative analyses of upstream regulatory sequences. Using the genome-reduced bacterium Mycoplasma pneumoniae, we show that ELM-seq has a large dynamic range and causes minimal toxicity. We use ELM-seq to determine key sequences (known and putatively novel) of promoter and untranslated regions that influence transcription and translation efficiency. Applying ELM-seq to other organisms will help us to further understand gene expression and guide synthetic biology.Quantitative analysis of how DNA sequence determines transcription and translation regulation is of interest to systems and synthetic biologists. Here the authors present ELM-seq, which uses Dam activity as reporter for high-throughput analysis of promoter and 5'-UTR regions.


Assuntos
Perfilação da Expressão Gênica/métodos , Mycoplasma pneumoniae/genética , Biossíntese de Proteínas , Transcrição Genética , Metilação de DNA , Escherichia coli/genética , Regulação da Expressão Gênica , Genes Reporter , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética
10.
J Phys Chem B ; 121(2): 365-378, 2017 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-28054779

RESUMO

Helicobacter pylori is a primitive Gram-negative bacterium that resides in the acidic environment of the human gastrointestinal tract, and some strains of this bacterium cause gastric ulcers and cancer. DNA methyltransferases (MTases) are promising drug targets for the treatment of cancer and other diseases that are also caused by epigenetic alternations of the genome. The N6-adenine-specific DNA MTase from H. pylori (M. Hpy N6mA) catalyzes the transfer of a methyl group from the cofactor S-adenosyl-l-methionine (AdoMet) to the flipped adenine of the substrate DNA. In this work, we report the sequence analyses, three-dimensional structure modeling, and molecular dynamics simulations of M. Hpy N6mA, when complexed with AdoMet as well as DNA. We analyzed the protein-DNA interactions prominently established by the flipped cytosine and the interactions between protein cofactors in the active site. The comparable orientation of AdoMet in both systems confirms that AdoMet is in a catalytically competent orientation in the bimolecular system that is retained upon DNA binding in the termolecular system of M. Hpy N6mA. In both systems, AdoMet is stabilized in the binding pocket by hydrogen bonding (Thr84, Glu99, Asp122, and Phe123) as well as van der Waals (Ile100, Phe160, Arg104, and Cys76) interactions. We propose that the contacts made by flipped adenine DA6 with Asn138 (N6 and N1 atom of DA6) and Pro139 (N6) and π-stacking interactions with Phe141 and Phe219 play an important role in the methylation mechanism at the N6 position in our N6mA model. Specific recognition of DNA is mediated by residues 143-155, 183-189, 212-220, 280-293, and 308-325. These findings are further supported by alanine scanning mutagenesis studies. The conserved residues in distantly related sequences of the small domain are important in DNA binding. Results reported here elucidate the sequence, structure, and binding features necessary for the recognition between cofactor AdoMet and substrate DNA by the vital epigenetic enzyme, M. Hpy N6mA.


Assuntos
DNA/metabolismo , S-Adenosilmetionina/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico , DNA/química , Helicobacter pylori , Ligações de Hidrogênio , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , S-Adenosilmetionina/química , Alinhamento de Sequência , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética
11.
Biochemistry ; 55(50): 6957-6960, 2016 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-27992993

RESUMO

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 DMSO and inhibited by glycerol, providing further corroboration that these proteins rely on distinct translocation mechanisms. The well-established similar effects of these osmolytes on bulk water, and their differential effects on macromolecule-associated waters, support our results and provide further evidence of the importance of water in interactions between macromolecules and their ligands.


Assuntos
DNA Bacteriano/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Proteínas de Escherichia coli/metabolismo , Osmose/fisiologia , Transporte Proteico/efeitos dos fármacos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Água/farmacologia , Sítios de Ligação , Crioprotetores/farmacologia , Metilação de DNA , DNA Bacteriano/química , Desoxirribonuclease EcoRI/química , Dimetil Sulfóxido/farmacologia , Proteínas de Escherichia coli/química , Glicerol/farmacologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Especificidade por Substrato
12.
Methods Mol Biol ; 1482: 49-66, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27557760

RESUMO

The specific binding of DNA-binding proteins to their cognate DNA motifs is a crucial step for gene expression control and chromatin organization in vivo. The development of methods for the identification of in vivo binding regions by, e.g. chromatin immunoprecipitation (ChIP) or DNA adenine methyltransferase identification (Dam-ID) added an additional level of qualitative information for data mining in systems biology or applications in synthetic biology. In this respect, the in vivo techniques outpaced methods for thorough characterization of protein-DNA interaction and, especially, of the binding motifs at single base-pair resolution. The elucidation of DNA-binding capacities of proteins is frequently done with methods such as yeast one-hybrid, electrophoretic mobility shift assay (EMSA) or systematic evolution of ligands by exponential enrichment (SELEX) that provide only qualitative binding information and are not suited for automation or high-throughput screening of several DNA motifs. Here, we describe the quantitative DNA-protein-Interaction-ELISA (qDPI-ELISA) protocol, which makes use of fluorescent fusion proteins and, hence, is faster and easier to handle than the classical DPI-ELISA. Although every DPI-ELISA experiment delivers quantitative information, the qDPI-ELISA has an increased consistency, as it does not depend on immunological detection. We demonstrate the high comparability between probes and different protein extracts in qDPI-ELISA experiments.


Assuntos
Imunoprecipitação da Cromatina/métodos , Proteínas de Ligação a DNA/química , DNA/genética , Sítios de Ligação , Cromatina/química , Cromatina/genética , Proteínas de Ligação a DNA/genética , Motivos de Nucleotídeos/genética , Ligação Proteica/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética
13.
Biochimie ; 128-129: 70-82, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27422119

RESUMO

EcoP15I DNA methyltransferase (M.EcoP15I) recognizes short asymmetric sequence, 5'-CAGCAG-3', and methylates the second adenine only on one strand of the double-stranded DNA (dsDNA). In vivo, this methylation is sufficient to protect the host DNA from cleavage by the cognate restriction endonuclease, R.EcoP15I, because of the stringent cleavage specificity requirements. Biochemical and structural characterization support the notion that purified M.EcoP15I exists and functions as dimer. However, the exact role of dimerization in M.EcoP15I reaction mechanism remains elusive. Here we engineered M.EcoP15I to a stable monomeric form and studied the role of dimerization in enzyme catalyzed methylation reaction. While the monomeric form binds single-stranded DNA (ssDNA) containing the recognition sequence it is unable to methylate it. Further we show that, while the monomeric form has AdoMet binding and Mg(2+) binding motifs intact, optimal dsDNA binding required for methylation is dependent on dimerization. Together, our biochemical data supports a unique subunit organization for M.EcoP15I to catalyze the methylation reaction.


Assuntos
Metilação de DNA , DNA/genética , Proteínas Recombinantes/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Sequência de Bases , Biocatálise , Dicroísmo Circular , DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Mutação , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , S-Adenosilmetionina/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Espectrometria de Fluorescência , Especificidade por Substrato
14.
Oncotarget ; 7(27): 40965-40977, 2016 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-27259995

RESUMO

DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria. However, the mechanism of sequence-specific recognition in N6-methyladenine modification remains elusive. M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes. Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 Å and 3.1 Å, respectively. The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme. Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity. Taken together, our data revealed the structural basis underlying DNA N6-adenine methyltransferase substrate promiscuity.


Assuntos
Helicobacter pylori/enzimologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Metilação de DNA , Helicobacter pylori/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Homologia de Sequência de Aminoácidos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Relação Estrutura-Atividade , Especificidade por Substrato
15.
J Vis Exp ; (107): e53620, 2016 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-26862720

RESUMO

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.


Assuntos
Mapeamento Cromossômico/métodos , Metilação de DNA , DNA/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Células 3T3 , Adenina/metabolismo , Animais , DNA/genética , DNA/metabolismo , Genômica/métodos , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de DNA/métodos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo
16.
Genesis ; 54(4): 151-9, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26845390

RESUMO

C. elegans has recently emerged as a valuable model to understand the link between nuclear organization and cell fate, by combining microscopy approaches, genome-wide mapping techniques with advanced genetics. Crucial to these analyses are techniques to determine the genome-wide interaction pattern of proteins with DNA. Chromatin immunoprecipitation has proven valuable but it requires considerable amounts of starting material. This is sometimes difficult to achieve, in particular for specific genotypes (balanced strains, different sexes, severe phenotypes…). As an alternative to ChIP, DNA adenine methyltransferase identification by sequencing (DamID-seq) was recently shown to be able to characterize binding sites in single mammalian cells. Additionally, DamID can be achieved for cell-type specific analysis by expressing Dam fusion proteins under tissue specific promoters in a controlled manner. In this report, we present a user-friendly pipeline to analyse DamID-seq data in C. elegans. Based upon this pipeline, we provide a comparative analysis of libraries generated with different starting material and discuss important library features. Moreover, we introduce an adaptation of an imaging based tool to visualize in vivo the cell-specific tridimensional binding pattern of any protein of interest.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , DNA de Helmintos/metabolismo , Análise de Sequência de DNA/métodos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Animais , Sítios de Ligação , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Metilação de DNA , Análise de Célula Única , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo
17.
J Biol Chem ; 291(12): 6124-33, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26797129

RESUMO

Bacterial HEMK2 homologs initially had been proposed to be involved in heme biogenesis or to function as adenine DNA methyltransferase. Later it was shown that this family of enzymes has protein glutamine methyltransferase activity, and they methylate the glutamine residue in the GGQ motif of ribosomal translation termination factors. The murine HEMK2 enzyme methylates Gln(185) of the eukaryotic translation termination factor eRF1. We have employed peptide array libraries to investigate the peptide sequence recognition specificity of murine HEMK2. Our data show that HEMK2 requires a GQX3R motif for methylation activity. In addition, amino acid preferences were observed between the -3 and +7 positions of the peptide substrate (considering the target glutamine as 0), including a preference for Ser, Arg, and Gly at the +1 and a preference for Arg at the +7 position. Based on our specificity profile, we identified several human proteins that contain putative HEMK2 methylation sites and show that HEMK2 methylates 58 novel peptide substrates. After cloning, expression, and purification of the corresponding protein domains, we confirmed methylation for 11 of them at the protein level. Transfected CHD5 (chromodomain helicase DNA-binding protein 5) and NUT (nuclear protein in testis) were also demonstrated to be methylated by HEMK2 in human HEK293 cells. Our data expand the range of proteins potentially subjected to glutamine methylation significantly, but further investigation will be required to understand the function of HEMK2-mediated methylation in proteins other than eRF1.


Assuntos
Processamento de Proteína Pós-Traducional , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , DNA Helicases/metabolismo , Células HEK293 , Humanos , Metilação , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/fisiologia , Especificidade por Substrato
18.
Biochimie ; 119: 60-7, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26475175

RESUMO

CcrM-related DNA-(adenine N6)-methyltransferases play very important roles in the biology of Caulobacter crescentus and other alpha-proteobacteria. These enzymes methylate GANTC sequences, but the molecular mechanism by which they recognize their target sequence is unknown. We carried out multiple sequence alignments and noticed that CcrM enzymes contain a conserved C-terminal domain (CTD) which is not present in other DNA-(adenine N6)-methyltransferases and we show here that deletion of this part abrogates catalytic activity and DNA binding of CcrM. A mutational study identified 7 conserved residues in the CTD (out of 13 tested), mutation of which led to a strong reduction in catalytic activity. All of these mutants showed altered DNA binding, but no change in AdoMet binding and secondary structure. Some mutants exhibited reduced DNA binding, but others showed an enhanced DNA binding. Moreover, we show that CcrM does not specifically bind to DNA containing GANTC sequences. Taken together, these findings suggest that the specific CcrM-DNA complex undergoes a conformational change, which is endergonic but essential for catalytic activity and this step is blocked by some of the mutations. Moreover, our data indicate that the CTD of CcrM is involved in DNA binding and recognition. This suggests that the CTD functions as target recognition domain of CcrM and, therefore, CcrM can be considered the first example of a δ-type DNA-(adenine N6)-methyltransferase identified so far.


Assuntos
Proteínas de Bactérias/química , Caulobacter crescentus/enzimologia , DNA/metabolismo , Modelos Moleculares , Estrutura Terciária de Proteína , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Códon de Terminação , Sequência Conservada , Metilação de DNA , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Especificidade por Substrato
19.
Chem Commun (Camb) ; 51(73): 13968-71, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26242378

RESUMO

We develop a sensitive fluorescence method for DNA methyltransferase (MTase) assay based on T7 RNA polymerase-mediated transcription amplification and duplex-specific nuclease (DSN)-assisted cyclic signal amplification. This method exhibits excellent specificity and high sensitivity with a detection limit of 0.015 U mL(-1), and it may be further applied for the screening of antimicrobial drugs.


Assuntos
RNA Polimerases Dirigidas por DNA/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Proteínas Virais/química , Antibacterianos/química , Fluorescência , Gentamicinas/química , Limite de Detecção , DNA Metiltransferases Sítio Específica (Adenina-Específica)/antagonistas & inibidores , Transcrição Genética
20.
Nat Commun ; 6: 7363, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-26067164

RESUMO

Type III R-M enzymes were identified >40 years ago and yet there is no structural information on these multisubunit enzymes. Here we report the structure of a Type III R-M system, consisting of the entire EcoP15I complex (Mod2Res1) bound to DNA. The structure suggests how ATP hydrolysis is coupled to long-range diffusion of a helicase on DNA, and how a dimeric methyltransferase functions to methylate only one of the two DNA strands. We show that the EcoP15I motor domains are specifically adapted to bind double-stranded DNA and to facilitate DNA sliding via a novel 'Pin' domain. We also uncover unexpected 'division of labour', where one Mod subunit recognizes DNA, while the other Mod subunit methylates the target adenine--a mechanism that may extend to adenine N6 RNA methylation in mammalian cells. Together the structure sheds new light on the mechanisms of both helicases and methyltransferases in DNA and RNA metabolism.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Helicases/metabolismo , Metilação de DNA , DNA/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Cristalização , Cristalografia por Raios X , Difusão , Escherichia coli , Hidrólise , Simulação de Acoplamento Molecular , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química
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