RESUMO
MspJI is a novel modification-dependent restriction endonuclease that cleaves at a fixed distance away from the modification site. Here, we present the biochemical characterization of several MspJI homologs, including FspEI, LpnPI, AspBHI, RlaI, and SgrTI. All of the enzymes specifically recognize cytosine C5 modification (methylation or hydroxymethylation) in DNA and cleave at a constant distance (N(12)/N(16)) away from the modified cytosine. Each displays its own sequence context preference, favoring different nucleotides flanking the modified cytosine. By cleaving on both sides of fully modified CpG sites, they allow the extraction of 32-base long fragments around the modified sites from the genomic DNA. These enzymes provide powerful tools for direct interrogation of the epigenome. For example, we show that RlaI, an enzyme that prefers (m)CWG but not (m)CpG sites, generates digestion patterns that differ between plant and mammalian genomic DNA, highlighting the difference between their epigenomic patterns. In addition, we demonstrate that deep sequencing of the digested DNA fragments generated from these enzymes provides a feasible method to map the modified sites in the genome. Altogether, the MspJI family of enzymes represent appealing tools of choice for method development in DNA epigenetic studies.
Assuntos
Enzimas de Restrição do DNA , Epigênese Genética , Epigenômica/métodos , Técnicas Genéticas , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Mapeamento Cromossômico/métodos , Biologia Computacional , DNA/química , DNA/genética , DNA/isolamento & purificação , Metilação de DNA , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , Biblioteca Gênica , Células HeLa , Humanos , Células Jurkat , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
A type IIG restriction endonuclease, RM.BpuSI from Bacillus pumilus, has been characterized and its X-ray crystal structure determined at 2.35Å resolution. The enzyme is comprised of an array of 5-folded domains that couple the enzyme's N-terminal endonuclease domain to its C-terminal target recognition and methylation activities. The REase domain contains a PD-x(15)-ExK motif, is closely superimposable against the FokI endonuclease domain, and coordinates a single metal ion. A helical bundle domain connects the endonuclease and methyltransferase (MTase) domains. The MTase domain is similar to the N6-adenine MTase M.TaqI, while the target recognition domain (TRD or specificity domain) resembles a truncated S subunit of Type I R-M system. A final structural domain, that may form additional DNA contacts, interrupts the TRD. DNA binding and cleavage must involve large movements of the endonuclease and TRD domains, that are probably tightly coordinated and coupled to target site methylation status.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/química , Sequência de Aminoácidos , Bacillus/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Clivagem do DNA , Metilases de Modificação do DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Mrr superfamily of homologous genes in microbial genomes restricts modified DNA in vivo. However, their biochemical properties in vitro have remained obscure. Here, we report the experimental characterization of MspJI, a remote homolog of Escherichia coli's Mrr and show it is a DNA modification-dependent restriction endonuclease. Our results suggest MspJI recognizes (m)CNNR (R = G/A) sites and cleaves DNA at fixed distances (N(12)/N(16)) away from the modified cytosine at the 3' side (or N(9)/N(13) from R). Besides 5-methylcytosine, MspJI also recognizes 5-hydroxymethylcytosine but is blocked by 5-glucosylhydroxymethylcytosine. Several other close homologs of MspJI show similar modification-dependent endonuclease activity and display substrate preferences different from MspJI. A unique feature of these modification-dependent enzymes is that they are able to extract small DNA fragments containing modified sites on genomic DNA, for example â¼32 bp around symmetrically methylated CG sites and â¼31 bp around methylated CNG sites. The digested fragments can be directly selected for high-throughput sequencing to map the location of the modification on the genomic DNA. The MspJI enzyme family, with their different recognition specificities and cleavage properties, provides a basis on which many future methods can build to decode the epigenomes of different organisms.