(27u) Understanding the Inherent Bias in the Activity of Mismatch Specific Exonuclease

Endonuclease NucS is a crucial enzyme involved in DNA repair and maintenance of genomic stability in bacteria and archaea. It is capable of detecting, cleaving, and repairing double-stranded breaks caused by exposure to ionizing radiation, chemicals, or other sources of DNA damage. Additionally, NucS enzymes can identify various DNA structures like branched DNA, Holliday junctions, and replication forks.

In a study by Nakae et al., the complex protein structure of NucS enzyme was analyzed, revealing that a dimer of the enzyme binds to double-stranded DNA at the cleavage site, causing mismatched bases to be flipped out of the binding site. This structure is like that of a restriction enzyme. Further Ishino et al. found that the NucS enzyme in Thermococcus kodakarensis can efficiently detect and cleave certain nucleotide mismatches, including G-T, G-G, T-T, T-C, and A-G, while having negligible activity towards C-C, A-C, and A-A mismatches. However, the reason for this preference for G and T over C and A nucleotides remains unclear, as well as whether this phenomenon is consistent among other NucS enzymes.

In our study, we aimed to investigate whether the preference for G and T nucleotides over C and A is a universal phenomenon among hyper-thermophilic NucS enzymes, and to gain a deeper understanding of the underlying reasons for this bias. To achieve this, we identified, expressed, purified, and characterized a diverse range of hyper-thermophilic NucS enzymes, and conducted substrate specificity assays to investigate potential differences in their activity. Furthermore, we analyzed the protein structure of these enzymes to elucidate how it may contribute to the observed preference.