Characterizing the effects and applications of Methylation dependent restriction endonucleases on genomic DNA

Abstract

Type IV Restriction Endonucleases are modification-based restriction enzymes which are typically methylation dependent. The Type IV restriction enzymes SauUSI and McrBC are ATP and GTP dependent respectively and specifically cleave DNA after recognising sequences containing methylated cytosine residues. Methylated Cytosine residues are commonly found in the genomic DNA of many organisms. In Prokaryotes, methylation of genomic DNA is common as part of Restriction-modification systems, and methylation of eukaryotic genomic DNA is an epigenetic modification that can influence the rates of transcription of genes.While SauUSI is a shredding enzyme that cleaves DNA in between two recognition sites into multiple small fragments, McrBC doesn’t shred DNA, but still cleaves methylated genomic DNA into small fragments due to it short, 2 base pair long recognition site. This ensures that treatment of methylated genomic DNA with either of these enzymes, or a combination of both, would cause the DNA to cleave into fragments of a short length. Here, we aimed to characterise and optimise the use of these Type IV restriction enzymes to selectively enrich unmethylated DNA in a mixture of methylated and unmethylated DNA for use in multiple applications, with the goal of developing a kit that could be used for this purpose in multiple applications in the future.