Oxidative stress in cells can lead to the accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized nucleotides such as 2'-deoxyribo-5-hydroxyuridin (HdU) and 2'-deoxyribo-5-hydroxymethyluridin (HMdU) can be inserted into DNA during replication and repair. HdU and HMdU have attracted particular interest because they have different effects on damaged-DNA processing enzymes that control the downstream effects of the lesions. Herein, we studied the chemically simulated translesion DNA synthesis (TLS) across the lesions formed by HdU or HMdU using microscale thermophoresis (MST). The thermodynamic changes associated with replication across HdU or HMdU show that the HdU paired with the mismatched deoxyribonucleoside triphosphates disturbs DNA duplexes considerably less than thymidine (dT) or HMdU. Moreover, we also demonstrate that TLS by DNA polymerases across the lesion derived from HdU was markedly less extensive and potentially more mutagenic than that across the lesion formed by HMdU. Thus, DNA polymerization by DNA polymerase η (polη), the exonuclease-deficient Klenow fragment of DNA polymerase I (KF-), and reverse transcriptase from human immunodeficiency virus type 1 (HIV-1 RT) across these pyrimidine lesions correlated with the different stabilization effects of the HdU and HMdU in DNA duplexes revealed by MST. The equilibrium thermodynamic data obtained by MST can explain the influence of the thermodynamic alterations on the ability of DNA polymerases to bypass lesions induced by oxidative products of pyrimidines. The results also highlighted the usefulness of MST in evaluating the impact of oxidative products of pyrimidines on the processing of these lesions by damaged DNA processing enzymes.

• Keywords
• 2’-deoxyribo-5-hydroxymethyl- uridin, 2’-deoxyribo-5-hydroxyuridin, DNA polymerases, microscale thermophoresis, oxidized nucleotides, translesion DNA synthesis,
• MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA biosynthesis   drug effects   MeSH
• HIV-1 MeSH
• Humans MeSH
• Mutagens chemistry   metabolism   pharmacology   MeSH
• DNA Repair MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress * MeSH
• Pentoxyl analogs & derivatives   chemistry   metabolism   pharmacology   MeSH
• DNA Damage MeSH
• Pyrimidines chemistry   metabolism   pharmacology   MeSH
• DNA Replication drug effects   MeSH
• Thermodynamics MeSH
• Uracil analogs & derivatives   chemistry   metabolism   pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 5-hydroxymethyluracil MeSH Browser
• 5-hydroxyuracil MeSH Browser
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH
• Mutagens MeSH
• Pentoxyl MeSH
• Pyrimidines MeSH
• Rad30 protein MeSH Browser
• Uracil MeSH