Abstract
Urea (Ua) is produced in DNA as the result of oxidative damage to thymine and guanine. It was previously reported that Klenow fragment (Kf) exo− incorporated dATP opposite Ua, and that DNA polymerase β was blocked by Ua. We report here the following nucleotide incorporations opposite Ua by various DNA polymerases: DNA polymerase α, dATP and dGTP (dATP > dGTP); DNA polymerase δ, dATP; DNA polymerase ζ, dATP; Kf exo−, dATP; Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), dGTP and dATP (dGTP > dATP); and DNA polymerase η, dCTP, dGTP, dATP, and dTTP (dCTP > dGTP > dATP > dTTP). DNA polymerases β and ε were blocked by Ua. Elongation by DNA polymerases δ and ζ stopped after inserting dATP opposite Ua. Importantly, the elongation efficiency to full-length beyond Ua using DNA polymerase η and Dpo4 were almost the same as that of natural DNA.Graphical abstract
Highlights
DNA damage is a major cause of cell death, mutations, cancer, neurological disease, and aging
Ua is reportedly repaired by many enzymes [3, 5, 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24], to date there have been only a few investigations into nucleotide incorporation and/or elongation past Ua by DNA polymerases, such as the blockage by Ua of DNA replication by T4 DNA polymerase [4, 25, 26], Klenow fragment (Kf) [4, 25,26,27,28], DNA polymerase I [25], human DNA polymerase β [28], Sequenase 2.0 [28], and AMV reverse transcriptase [28]
Under the reaction condition in which DNA polymerase α inserted only dCTP opposite guanine (Fig. 2A, lanes 1–4), dATP and dGTP were inserted opposite Ua, and dTTP and dCTP were not inserted (Fig. 2A, lanes 6–9)
Summary
DNA damage is a major cause of cell death, mutations, cancer, neurological disease, and aging. Ua is reportedly repaired by many enzymes [3, 5, 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24], to date there have been only a few investigations into nucleotide incorporation and/or elongation past Ua by DNA polymerases, such as the blockage by Ua of DNA replication by T4 DNA polymerase [4, 25, 26], Klenow fragment (Kf) [4, 25,26,27,28], DNA polymerase I [25], human DNA polymerase β [28], Sequenase 2.0 [28], and AMV reverse transcriptase [28]. Nucleotide incorporations opposite Ua and translesion synthesis across Ua by DNA polymerases α, δ, ε, η, ζ and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) have not
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