TY - JOUR
T1 - Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing
AU - Baljinnyam, Tuvshintugs
AU - Conrad, James W.
AU - Sowers, Mark L.
AU - Chang-Gu, Bruce
AU - Herring, Jason L.
AU - Hackfeld, Linda C.
AU - Zhang, Kangling
AU - Sowers, Lawrence C.
N1 - Funding Information:
This work was funded in part by a grant from the NIH NCI R01CA228085, the John Sealy Distinguished chair in Cancer Biology, the Keating Endowment, and the NSF EFRI1933321. J.W.C. was funded in part by the Computational Cancer Biology Training Program fellowship (CCBTP) Grant No. RP170593. M.L.S. and B.C. were supported in part by the UTMB physician-scientist training program. We thank Ellie Cherryhomes for improving the visualization of the manuscript. We thank the editor and reviewers for their helpful suggestions that improved the quality of the manuscript.
Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.
PY - 2023/2/20
Y1 - 2023/2/20
N2 - Recently, we constructed a hybrid thymine DNA glycosylase (hyTDG) by linking a 29-amino acid sequence from the human thymine DNA glycosylase with the catalytic domain of DNA mismatch glycosylase (MIG) from M. thermoautotrophicum, increasing the overall activity of the glycosylase. Previously, it was shown that a tyrosine to lysine (Y126K) mutation in the catalytic site of MIG could convert the glycosylase activity to a lyase activity. We made the corresponding mutation to our hyTDG to create a hyTDG-lyase (Y163K). Here, we report that the hybrid mutant has robust lyase activity, has activity over a broad temperature range, and is active under multiple buffer conditions. The hyTDG-lyase cleaves an abasic site similar to endonuclease III (Endo III). In the presence of β-mercaptoethanol (β-ME), the abasic site unsaturated aldehyde forms a β-ME adduct. The hyTDG-lyase maintains its preference for cleaving opposite G, as with the hyTDG glycosylase, and the hyTDG-lyase and hyTDG glycosylase can function in tandem to cleave T:G mismatches. The hyTDG-lyase described here should be a valuable tool in studies examining DNA damage and repair. Future studies will utilize these enzymes to quantify T:G mispairs in cells, tissues, and genomic DNA using next-generation sequencing.
AB - Recently, we constructed a hybrid thymine DNA glycosylase (hyTDG) by linking a 29-amino acid sequence from the human thymine DNA glycosylase with the catalytic domain of DNA mismatch glycosylase (MIG) from M. thermoautotrophicum, increasing the overall activity of the glycosylase. Previously, it was shown that a tyrosine to lysine (Y126K) mutation in the catalytic site of MIG could convert the glycosylase activity to a lyase activity. We made the corresponding mutation to our hyTDG to create a hyTDG-lyase (Y163K). Here, we report that the hybrid mutant has robust lyase activity, has activity over a broad temperature range, and is active under multiple buffer conditions. The hyTDG-lyase cleaves an abasic site similar to endonuclease III (Endo III). In the presence of β-mercaptoethanol (β-ME), the abasic site unsaturated aldehyde forms a β-ME adduct. The hyTDG-lyase maintains its preference for cleaving opposite G, as with the hyTDG glycosylase, and the hyTDG-lyase and hyTDG glycosylase can function in tandem to cleave T:G mismatches. The hyTDG-lyase described here should be a valuable tool in studies examining DNA damage and repair. Future studies will utilize these enzymes to quantify T:G mispairs in cells, tissues, and genomic DNA using next-generation sequencing.
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U2 - 10.1021/acs.chemrestox.2c00172
DO - 10.1021/acs.chemrestox.2c00172
M3 - Article
C2 - 36647573
AN - SCOPUS:85146548303
SN - 0893-228X
VL - 36
SP - 162
EP - 176
JO - Chemical Research in Toxicology
JF - Chemical Research in Toxicology
IS - 2
ER -