(164m) Thermostable Enzymes for Error Correction of DNA

The ability to synthesize accurate DNA is important for advancements in synthetic biology and biotechnology. Contrary to DNA replication in vivo, in vitro DNA synthesis lacks DNA repair mechanism and pathways to detect and repair mismatches. Enzymatic error correction for DNA synthesis is a cost-effective method of reducing errors in synthetically generate DNA. DNA-based error correction enzymes are known to detect specific base-pair mismatches and work by cleaving both DNA strands at 3’ end of the mismatched site. Currently, these error correction enzymes are highly accurate but lack thermal stability and demonstrate poor enzyme kinetics. Thermophiles were found to have lower mutation rate as compared to mesophiles and therefore, surveyor endonucleases in thermophilic bacteria were explored and characterized for functioning error correction activity and higher temperature stabilities. This specific study focuses on an error correction enzyme from Thermococcus Barophilus which is abbreviated as enzyme TbaMP. This enzyme was sequence optimized, cloned, expressed in Escherichia coli, and purified for activity and thermostability tests. Activity assay demonstrated that the enzyme TbaMP had superior G/C mismatch detection and correction ability as compared to the commercial standard. Ultimately, this data supports the efficacy of thermophilic error correction enzymes and supports further investigation and application of this enzyme in DNA error correction.