Mass spectrometry-based assays for assessing replicative bypass and repair of DNA alkylation in cells

Abstract

Endogenous metabolism and environmental exposure can give rise to DNA alkylation, which can elicit deleterious biological consequences. In the search for reliable and quantitative analytical methods to elucidate the impact of DNA alkylation on the flow of genetic information, mass spectrometry (MS) has attracted increasing attention, owing to its unambiguous determination of molecular mass. The MS-based assays obviate conventional colony-picking methods and Sanger sequencing procedures, and retained the high sensitivity of postlabeling methods. With the help of the CRISPR/Cas9 gene editing method, MS-based assays showed high potential in studying individual functions of repair proteins and translesion synthesis (TLS) polymerases in DNA replication. In this mini-review, we have summarized the development of MS-based competitive and replicative adduct bypass (CRAB) assays and their recent applications in assessing the impact of alkylation on DNA replication. With further development of MS instruments for high resolving power and high throughput, these assays should be generally applicable and efficient in quantitative measurement of the biological consequences and repair of other DNA lesions.

Fig. 1. (a) Schematic illustration of experimental procedures of CRAB in E. coli cells. (b) Sequential restriction enzyme digestion for MS-based replication assay in E. coli cells (top) and a representative MS spectrum (bottom) showing the T → C mutation elicited by O⁴-methylthymidine (O⁴-MedT). (c) Sequential restriction enzyme digestion for the selective labeling of the strand initially bearing the lesion or the complementary strand, and the representative images of PAGE gel. The ‘M’ labeled in blue and ‘N’ labeled in orange indicate the nucleotide at the lesion-situated stand and lesion-complementary strand after replication, respectively. Reproduced from ref. with permission from Oxford, copyright 2015.

Fig. 2. Experimental procedures for MS-based replication assay in mammalian cells. The C/C mismatch site, the lesion site, and the incorporated nucleotides at the lesion site and opposite lesion site are marked in green, red, blue, and orange. The SfaNI and NcoI sites are highlighted in bold.

Fig. 3. Chemical structures of the examined alkyl groups (a) and replicative bypass efficiencies of O²-alkyldT and O⁴-alkyldG lesions (b and c) in E. coli cells. Reproduced from ref. with permission from Oxford, copyright 2015 and ref. with permission from Elsevier, copyright 2018.

Fig. 4. Replicative bypass efficiencies of O⁴-alkyldT (a) and O⁶-alkyl-dG lesions (b) in mammalian cells. Reproduced from ref. with permission from Oxford, copyright 2016 and ref. with permission from Elsevier, copyright 2019.

Fig. 5. Replicative bypass efficiencies of O²-alkyldT (a) and N²-alkyl-dG lesions (b) in mammalian cells. Reproduced from ref. with permission from Elsevier, copyright 2018 and ref. with permission from American Chemical Society, copyright 2019.

Fig. 6. S _p and R_p diastereomers of alkyl phosphotriester residues in DNA. The ‘X’ indicates the substituted alkyl group.

Jiaxian Li

Zhihai Hu

Dandan Liu

Pengcheng Wang