pubmed.ncbi.nlm.nih.gov

Biochemical analysis of H2O2-induced mutation spectra revealed that multiple damages were involved in the mutational process - PubMed

Biochemical analysis of H2O2-induced mutation spectra revealed that multiple damages were involved in the mutational process

Tomohiko Sugiyama et al. DNA Repair (Amst). 2024 Feb.

Abstract

Reactive oxygen species (ROS) are a major threat to genomic integrity and believed to be one of the etiologies of cancers. Here we developed a cell-free system to analyze ROS-induced mutagenesis, in which DNA was exposed to H2O2 and then subjected to translesion DNA synthesis by various DNA polymerases. Then, frequencies of mutations on the DNA products were determined by using next-generation sequencing technology. The majority of observed mutations were either C>A or G>A, caused by dAMP insertion at G and C residues, respectively. These mutations showed similar spectra to COSMIC cancer mutational signature 18 and 36, which are proposed to be caused by ROS. The in vitro mutations can be produced by replicative DNA polymerases (yeast DNA polymerase δ and ε), suggesting that ordinary DNA replication is sufficient to produce them. Very little G>A mutation was observed immediately after exposure to H2O2, but the frequency was increased during the 24 h after the ROS was removed, indicating that the initial oxidation product of cytosine needs to be maturated into a mutagenic lesion. Glycosylase-sensitivities of these mutations suggest that the C>A were made on 8-oxoguanine or Fapy-guanine, and that G>A were most likely made on 5-hydroxycytosine modification.

Keywords: 5-hydroxycytosine; 8-oxoguanine; Cancer mutational signature; DNA Polymerase delta; In vitro mutagenesis; Reactive oxygen species.

Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.

PubMed Disclaimer

Conflict of interest statement

Declaration of Competing Interest The authors declare that there are no conflict of interest.

Figures

Fig. 1.
Fig. 1.

(A) Illustration of DNA damaging and primer-extension. A 32P-labeled primer (36-nt) was annealed with H2O2-treated DNA and extended by yPol δ (T = 0). When indicated, another polymerase (2nd Pol) was added at T=15 min. See Materials and Methods for details of the procedure. (B) Time-course of H2O2-exposure. A 100-nt ssDNA (template A) was incubated with 5 mM H2O2 for indicated periods and subjected to the primer-extension assay with yPol δ. (C) Primer-extension was carried out using yPol δ and the indicated 2nd Pol (‘η’ = hPol η, ‘k’ = hPol k). Samples were withdrawn from the reactions at indicated times ‘T’ and analyzed by gel electrophoresis. (D). Similar experiments were repeated using indicated 2nd Pols (‘ι’ = hPol ι and ‘ζ’ = yPol ζ) and relative amounts of full-length products in the presence/the absence of the 2nd Pols were plotted. (E-F) Illustration of H2O2-induced mutation assay. NGS primer containing an A-adaptor (red box) and a barcode (BC) was annealed with H2O2-treated or -untreated template DNA and extended by polymerase. (G-J) Examples of H2O2-induced mutation analysis. Template A was exposed to 5 mM of H2O2 for 8 hrs and subjected to the primer-extension by indicated polymerases. Then single-nucleotide substitution frequencies were quantified and mapped on the template sequence.

Fig. 2.
Fig. 2.

Primer-extension on the template that contains an 8-oxoG at the defined position. (A). Illustration of the system. Exact structure of the template is shown in Supplementary Fig. S1B. (B) Results of the primer extension by yPol δ and indicated 2nd Pol. (C) Nucleotides that were inserted at 8-oxoG in the single polymerase reactions. Experiment was repeated twice and mean with variation are shown.

Fig. 3.
Fig. 3.

(A-C) H2O2-induced mutation frequencies that were produced by yPol δ (A), yPol δ + hPol η (B), and yPol δ + hPol κ (C) after subtracting background. Each dot represents the mutation frequency at particular base of the templates. Bar is mean with 95% IC. Number of template bases (n) are A = 88, C = 90, G = 88, and T = 84. (D-E) Scatter plots of mutation frequencies that were produced on the same H2O2-damaged DNA with different polymerases (n values are shown in the graph).

Fig. 4.
Fig. 4.. Post-exposure time-course.

(A) Reaction scheme. Template was exposed to 200 mM of H2O2 for 30 min and then purified twice through G25 spin-columns that were preequilibrated with 30 mM Na-Phosphate buffer (pH 7.5). Then the DNA was incubated at 37°C to facilitate maturation of lesions. Small aliquots were withdrawn at indicated times after removal of H2O2 and immediately subjected to the primer-extension by yPol δ for NGS sequencing. (B-C) The post-exposure time-course of C>A (n = 88) and C>G (n= 88) and G>A (n = 90) mutations (mean +/− SEM). “Controls” in each panel are the results of the same experiment except that the spin-column purification was carried out immediately after H2O2 was added. (D) Reaction scheme of simple time course of H2O2-exposure. Template was incubated with 10 mM of H2O2 for indicated periods and aliquots were withdrawn, purified, and subjected to the primer-extension by yPol δ, and the mutation frequencies were determined (E).

Fig. 5.
Fig. 5.

(A) Reaction scheme of dsDNA damaging and mutation assay. The dsDNA substrates were incubated with 10 mM H2O2 for 8 hours at 37°C and then purified with G-25 spin-column, and further incubated for 24 hours at 37°C. The DNA was heated and reannealed with a NGS primer and 10x excess competitor to sequester the top strand. Then the primer-extension and NGS analysis were carried out. (B) An example to explain how in vitro mutation spectrum was obtained. In this example, strand extension occurs from left to right on the damaged template. During the TLS, two misincorporations (G>A in blue and C>T in green) occur on the extending strand. These changes are complementary to each other including the sequence context, making them indistinguishable by genome sequencing. Since our in vitro approach separately quantifies such complementary mutations on the extending strand, we show them at the same position on the graph by a blue bar (purine substitution) and a green bar (pyrimidine substitution). (C) Mutation spectra on the damaged dsDNA by indicated polymerases (mean +SD). (D) Cosmic signatures (SBS) 18, 36, and 29 are normalized by the appearances of trinucleotide sequences in human genome. (E) Cosine similarities between the in vitro spectra and the cancer signatures.

Fig. 6.
Fig. 6.

Effects of various glycosylases on the in vitro mutation spectrum produced on dsDNA that was treated with H2O2. (A) The mutation spectra were obtained from the same experiment as in Fig. 5C (yPol δ-only) except that the damaged dsDNA was treated with indicated glycosylases before primer-extension by yPol δ. (B) Effects of the glycosylases on C>A and G>A mutation frequencies were obtained as the ratios of mutation frequencies in the presence/absence of the glycosylase at individual template G and C bases. To avoid excessive data fluctuations, the ratios were calculated only at the bases that showed 0.1% or higher mutation frequencies in the absence of the glycosylase (n values are shown in the Figure). (C) Major oxidation products of G and C residues that can be removed by the indicated glycosylases. References: a (40–42), b (43,44), c (45,46), d (43,44,47–50), e (50,51), f (52–54), g (43).

Fig. 7.
Fig. 7.

Working model of ROS-induced mutational mechanism. See text for explanation.

Similar articles

Cited by

References

    1. Cadet J and Wagner JR. DNA base damage by reactive oxygen species, oxidizing agents, and UV radiation. Cold Spring Harbor perspectives in biology, 5 (2013). 10.1101/cshperspect.a012559. - DOI - PMC - PubMed
    1. Bjelland S and Seeberg E. Mutagenicity, toxicity and repair of DNA base damage induced by oxidation. Mutat Res, 531 (2003) 37–80. - PubMed
    1. Dizdaroglu M. Oxidative damage to DNA in mammalian chromatin. Mutat Res, 275 (1992) 331–342. 10.1016/0921-8734(92)90036-o. - DOI - PubMed
    1. Yamaguchi R, Hirano T, Asami S, Chung MH, Sugita A and Kasai H. Increased 8-hydroxyguanine levels in DNA and its repair activity in rat kidney after administration of a renal carcinogen, ferric nitrilotriacetate. Carcinogenesis, 17 (1996) 2419–2422. 10.1093/carcin/17.11.2419. - DOI - PubMed
    1. Gedik CM, Collins A and Escodd. Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study. FASEB J, 19 (2005) 82–84. 10.1096/fj.04-1767fje. - DOI - PubMed

MeSH terms

Substances

LinkOut - more resources