RESUMO
DNMT3A/3L heterotetramers contain two active centers binding CpG sites at 12 bp distance, however their interaction with DNA not containing this feature is unclear. Using randomized substrates, we observed preferential co-methylation of CpG sites with 6, 9 and 12 bp spacing by DNMT3A and DNMT3A/3L. Co-methylation was favored by AT bases between the 12 bp spaced CpG sites consistent with their increased bending flexibility. SFM analyses of DNMT3A/3L complexes bound to CpG sites with 12 bp spacing revealed either single heterotetramers inducing 40° DNA bending as observed in the X-ray structure, or two heterotetramers bound side-by-side to the DNA yielding 80° bending. SFM data of DNMT3A/3L bound to CpG sites spaced by 6 and 9 bp revealed binding of two heterotetramers and 100° DNA bending. Modeling showed that for 6 bp distance between CpG sites, two DNMT3A/3L heterotetramers could bind side-by-side on the DNA similarly as for 12 bp distance, but with each CpG bound by a different heterotetramer. For 9 bp spacing our model invokes a tetramer swap of the bound DNA. These additional DNA interaction modes explain how DNMT3A and DNMT3A/3L overcome their structural preference for CpG sites with 12 bp spacing during the methylation of natural DNA.
Assuntos
Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , DNA/genética , Sítios de Ligação/genética , DNA/ultraestrutura , DNA (Citosina-5-)-Metiltransferases/ultraestrutura , DNA Metiltransferase 3A , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/ultraestrutura , Humanos , Domínios Proteicos/genéticaRESUMO
DNA methylation and H3K9me are hallmarks of heterochromatin in plants and mammals, and are successfully maintained across generations. The biochemical and structural basis for this maintenance is poorly understood. The maintenance DNA methyltransferase from Zea mays, ZMET2, recognizes dimethylation of H3K9 via a chromodomain (CD) and a bromo adjacent homology (BAH) domain, which flank the catalytic domain. Here, we show that dinucleosomes are the preferred ZMET2 substrate, with DNA methylation preferentially targeted to linker DNA. Electron microscopy shows one ZMET2 molecule bridging two nucleosomes within a dinucleosome. We find that the CD stabilizes binding, whereas the BAH domain enables allosteric activation by the H3K9me mark. ZMET2 further couples recognition of H3K9me to an increase in the specificity for hemimethylated versus unmethylated DNA. We propose a model in which synergistic coupling between recognition of nucleosome spacing, H3K9 methylation, and DNA modification allows ZMET2 to maintain DNA methylation in heterochromatin with high fidelity.
Assuntos
Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Nucleossomos/enzimologia , Proteínas de Plantas/metabolismo , Animais , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/ultraestrutura , Ativação Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Microscopia Eletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/ultraestrutura , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestrutura , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
The DNA methyltransferases, M.HhaI and M.TaqI, and catechol O-methyl-transferase (COMT) catalyze the transfer of a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to carbon-5 of cytosine, to nitrogen-6 of adenine, and to a hydroxyl group of catechol, respectively. The catalytic domains of the bilobal proteins, M.HhaI and M.TaqI, and the entire single domain of COMT have similar folding with an alpha/beta structure containing a mixed central beta-sheet. The functional residues are located in equivalent regions at the carboxyl ends of the parallel beta-strands. The cofactor binding sites are almost identical and the essential catalytic amino acids coincide. The comparable protein folding and the existence of equivalent amino acids in similar secondary and tertiary positions indicate that many (if not all) AdoMet-dependent methyltransferases have a common catalytic domain structure. This permits tertiary structure prediction of other DNA, RNA, protein, and small-molecule AdoMet-dependent methyltransferases from their amino acid sequences.
Assuntos
Metilases de Modificação do DNA/química , Metiltransferases/química , S-Adenosilmetionina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catecol O-Metiltransferase/química , Catecol O-Metiltransferase/ultraestrutura , Metilases de Modificação do DNA/ultraestrutura , Metiltransferases/ultraestrutura , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
The following ligands were used to study sequence specific recognition of duplex DNA by electron microscopic techniques: methyltransferases BspR1 and EcoR124 (recognition sequences GGCC and GAAN7RTCG, respectively), a biotinylated deoxyoligonucleotide 5'-CTCTCTCTCTCTCT-3' capable of forming triplex DNA, and PNA oligomer H-T10-LysNH2. For each ligand the best conditions for electron microscopic (EM) detection of stable specific complex formation were determined. It was demonstrated that EM allowed us to determine the position of the individual target site with an error of 15-20 bp, the relative affinities for individual target sites and kinetic parameters of the binding. These results open new possibilities for EM investigations of sequence-specific interactions with a wide range of other ligands of a similar nature. They also imply that a wide range of different sequences can be unambiguously and precisely mapped by EM and greatly extend the scope of EM applications for physical mapping of genomic DNA.
