The MspJI family of restriction endonucleases - characterization, mechanism and application
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MspJI is a novel modification-dependent restriction endonuclease that specifically recognizes cytosine C5 modification (methylation or hydroxymethylation) in DNA and cleaves at a constant distance (N12/N16) away from the modified cytosine site. The biochemical characterization of MspJI and several homologs, including FspEI, LpnPI, AspBHI, RiaI, and SgrTI is presented in this thesis. Each displays its own sequence context preference, favoring different nucleotides flanking the modified cytosine. Despite the difference in the recognition sequence, the MspJI family enzymes display similarities in their other properties, such as the distance from the modified cytosine to the cleavage site, stimulation of cleavage by a short DNA oligonucleotide containing the recognition site and the ability to produce a double strand break in molecules with a single recognition site. Here, the possibility of multimerization as part of the mechanism by which these enzymes are able to display those properties is discussed. In addition, by cleaving on both sides of fully modified CpG sites, these enzymes allow the extraction of 32-base long fragments around the modified sites from the genomic DNA, providing powerful tools for direct interrogation of the epigenome. For example, Rial , an enzyme that prefers mcwG but not mCpG sites, generates digestion patterns that differ between plant and mammalian genomic DNA, highlighting the difference between their epigenomic patterns. Furthermore, deep sequencing of the digested DNA fragments generated from these enzymes provides a feasible method to map the modified sites in the genome.
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