Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing

• DOI: 10.1021/acs.chemrestox.2c00172
• Publication Date: 2023-01-17T07:11:56Z
• Authors: Tuvshintugs Balji...; James W. Conrad; Mark L. Sowers; Bruce Chang-Gu; Jason L. Herring; Linda C. Hackfeld; Kangling Zhang; Lawrence C. Sowers
• Keywords: Lyase; AP endonuclease; MUTYH

Recently, we constructed a hybrid thymine DNA glycosylase (hyTDG) by linking 29-amino acid sequence from the human with catalytic domain of mismatch (MIG) M. thermoautotrophicum, increasing overall activity glycosylase. Previously, it was shown that tyrosine to lysine (Y126K) mutation in site MIG could convert lyase activity. We made corresponding our hyTDG create hyTDG-lyase (Y163K). Here, report mutant has robust activity, over broad temperature range, and is active under multiple buffer conditions. The cleaves an abasic similar endonuclease III (Endo III). In presence β-mercaptoethanol (β-ME), unsaturated aldehyde forms β-ME adduct. maintains its preference for cleaving opposite G, as glycosylase, can function tandem cleave T:G mismatches. described here should be valuable tool studies examining damage repair. Future will utilize these enzymes quantify mispairs cells, tissues, genomic using next-generation sequencing.