Mass Spectrometry-Based Quantitative Strategies for Assessing the Biological Consequences and Repair of DNA Adducts

Abstract

The genetic integrity of living organisms is constantly threatened by environmental and endogenous sources of DNA damaging agents that can induce a plethora of chemically modified DNA lesions. Unrepaired DNA lesions may elicit cytotoxic and mutagenic effects and contribute to the development of human diseases including cancer and neurodegeneration. Understanding the deleterious outcomes of DNA damage necessitates the investigation about the effects of DNA adducts on the efficiency and fidelity of DNA replication and transcription. Conventional methods for measuring lesion-induced replicative or transcriptional alterations often require time-consuming colony screening and DNA sequencing procedures. Recently, a series of mass spectrometry (MS)-based strategies have been developed in our laboratory as an efficient platform for qualitative and quantitative analyses of the changes in genetic information induced by DNA adducts during DNA replication and transcription. During the past few years, we have successfully used these MS-based methods for assessing the replicative or transcriptional blocking and miscoding properties of more than 30 distinct DNA adducts. When combined with genetic manipulation, these methods have also been successfully employed for revealing the roles of various DNA repair proteins or translesion synthesis DNA polymerases (Pols) in modulating the adverse effects of DNA lesions on transcription or replication in mammalian and bacterial cells. For instance, we found that Escherichia coli Pol IV and its mammalian ortholog (i.e., Pol κ) are required for error-free bypass of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) in cells. We also found that the N(2)-CEdG lesions strongly inhibit DNA transcription and they are repaired by transcription-coupled nucleotide excision repair in mammalian cells. In this Account, we focus on the development of MS-based approaches for determining the effects of DNA adducts on DNA replication and transcription, where liquid chromatography-tandem mass spectrometry is employed for the identification, and sometimes quantification, of the progeny products arising from the replication or transcription of lesion-bearing DNA substrates in vitro and in mammalian cells. We also highlight their applications to lesion bypass, mutagenesis, and repair studies of three representative types of DNA lesions, that is, the methylglyoxal-induced N(2)-CEdG, oxidatively induced 8,5'-cyclopurine-2'-deoxynucleosides, and regioisomeric alkylated thymidine lesions. Specially, we discuss the similar and distinct effects of the minor-groove DNA lesions including N(2)-CEdG and O(2)-alkylated thymidine lesions, as well as the major-groove O(4)-alkylated thymidine lesions on DNA replication and transcription machinery. For example, we found that the addition of an alkyl group to the O(4) position of thymine may facilitate its preferential pairing with guanine and thus induce exclusively the misincorporation of guanine nucleotide opposite the lesion, whereas alkylation of thymine at the O(2) position may render the nucleobase unfavorable in pairing with any of the canonical nucleobases and thus exhibit promiscuous miscoding properties during DNA replication and transcription. The MS-based strategies described herein should be generally applicable for quantitative measurement of the biological consequences and repair of other DNA lesions in vitro and in cells.

Figure 1

Experimental procedures for two MS-based in vitro replication assays (a, b), and representative selected-ion chromatograms revealing the distributions of SacI-treated primer extension products from human Pol η-mediated replication of O²-EtdT-bearing substrate in vitro (c) The uracil site is underlined, and the SacI site is highlighted in bold. ‘X’ indicates a lesion or unmodified base. Adapted from ref. 25. Copyright 2013 American Chemical Society.

Figure 2

A schematic diagram illustrating the procedures for the construction of lesion-bearing M13 genome (a) and an outline of MS-based replication assay in E. coli cells (b). The BbsI and MluCI sites are highlighted in bold. ‘X’ indicates a lesion or unmodified base.

Figure 3

LC-MS and MS/MS for monitoring the identities of restriction fragments arising from the replication of O²-EtdT-bearing plasmid in E. coli cells. (a) High resolution “ultra zoom-scan” ESI-MS revealed the presence of the [M-3H]³⁻ ions of 10-mer restriction fragment d(AATTATAGCN), with ‘N’ being A (wild-type) or C/G/T (A→C/G/T mutation). (b) A representative product-ion spectrum (MS/MS) of the [M-3H]³⁻ ion (m/z 1002.2) of the mutant sequence d(AATTATAGCC). Shown above the spectrum is a scheme summarizing the observed [a_n − Base] and w_n fragment ions, and nomenclature follows that described previously. Adapted with permission from ref. 19. Copyright 2014 Oxford University Press.

Figure 4

Experimental procedures for two MS-based replication assays in mammalian cells (a, b). The A/A and C/C mismatch sites are underlined, and the SacI, FspI, SfaNI and NcoI sites are highlighted in bold.

Figure 5

Outline of the CTAB assay. ‘X’ indicates a lesion or unmodified base , which is located on the transcribed strand of the TurboGFP gene downstream of the CMV and T7 promoters. The arrowheads indicate the +1 transcription start sites of CMV and T7 promoters. Adapted with permission from ref. 23. Copyright 2015 Oxford University Press.

Figure 6

Chemical structures of representative DNA lesions discussed in this Account.

Figure 7

Replicative bypass efficiencies (a) and mutagenic potentials (b) of N3-, O²- and O⁴-EtdT in E. coli cells. Adapted with permission from ref. 19. Copyright 2014 Oxford University Press.

Figure 8

Chemical structures of the examined alkyl groups (a), and replicative bypass efficiencies of O²- and O⁴-alkyldT lesions (b, c) in E. coli cells. Adapted with permission from refs. 20 and 29. Copyright 2014 and 2015 Oxford University Press.

Figure 9

Bypass efficiencies (a, b) and mutagenic potentials (c) of N²-CEdG lesions during replication and transcription in mammalian cells. The 293T cell line is a derivative of human embryonic kidney 293 cell line and contains the SV40 T-antigen. GM04429 and GM00637 are NER-deficient (lacking XPA) and repair-proficient human cell lines, respectively. N²-CEdG lesions are not mutagenic during replication in wild-type mammalian cells, but they can induce G→T and G→A mutations in mammalian cells that are deficient in Pol κ or Pol ι. Adapted with permission from refs. 11 and 15. Copyright 2012 Nature Publishing Group (Figure 9b). Figure 9a was adapted from the data originally published in J. Biol. Chem. Yuan, B. et al. The roles of DNA polymerases κ and ι in the error-free bypass of N²-carboxyalkyl-2′-deoxyguanosine lesions in mammalian cells. J. Biol. Chem. 2011; Vol 286: 17503-17511. © the American Society for Biochemistry and Molecular Biology.

Figure 10

Bypass efficiencies (a, b) and mutagenic potentials (c) of S-cdA and S-cdG during replication and transcription in mammalian cells. Adapted with permission from refs. 11 and 17. Copyright 2012 Nature Publishing Group (Figure 10b). Figure 10a was adapted from the data originally published in J. Biol. Chem. You, C. et al. Translesion synthesis of 8,5'-cyclopurine-2'-deoxynucleosides by DNA polymerases η, ι, and ζ. J. Biol. Chem. 2013; Vol 288: 28548-28556. © the American Society for Biochemistry and Molecular Biology.