Our laboratories published LC-MS methods for analysis of oligonucleotide primer extension products in 2005, along with our first X-ray crystal structure of the translesion DNA polymerase Sulfolobus solfataricus Dpo4 copying past the adduct 1,N2-etheno (ε) G. Subsequently we obtained crystal structures of Dpo4 mispairs with N2,3-εG, O6-MeG, O6-BzG, 8-oxoG, N2,N2-Me2G, and N2-Me naphthalene. These structures revealed a number of aspects of Dpo4, along with a study of kinetics with tryptophan mutants and proton-deuterium exchange work. We have solved a number of structures of human translesion DNA polymerases with DNA adducts, including: (a) pol k and 8-oxoG; (b) pol ι with O6-MeG and N2,3-εG; and (c) pol η with 8-oxoG, O6-MeG, 1,N6-εA, 1,N2-εG, an abasic (AP) site, and a ribonucleotide. Some interesting findings included the similarity of AP sites and 1,N6-εA in driving the insertion of purines and frameshifts. Although pol η has steric gate residues, it has activity with ribo sugars and is also able to copy ribonucleotide templates, i.e. reverse transcriptase activity. Current interests also involve DNA-protein crosslinks, although crystal structures are not yet in hand.

Sixteen years of catching DNA polymerases in the act of mistakes: What have we learned about DNA adducts?

ACS Fall 2021

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