Mechanism of DNA alkylation-induced transcriptional stalling, lesion bypass, and mutagenesis

Abstract

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, interfere with the efficiency and accuracy of DNA replication and transcription. However, the molecular mechanisms of DNA alkylation-induced transcriptional stalling and mutagenesis remain unknown. In this study, we systematically investigated how RNA polymerase II (pol II) recognizes and bypasses regioisomeric O²-, N3-, and O⁴-ethylthymidine (O²-, N3-, and O⁴-EtdT) lesions. We observed distinct pol II stalling profiles for the three regioisomeric EtdT lesions. Intriguingly, pol II stalling at O²-EtdT and N3-EtdT sites is exacerbated by TFIIS-stimulated proofreading activity. Assessment for the impact of the EtdT lesions on individual fidelity checkpoints provided further mechanistic insights, where the transcriptional lesion bypass routes for the three EtdT lesions are controlled by distinct fidelity checkpoints. The error-free transcriptional lesion bypass route is strongly favored for the minor-groove O²-EtdT lesion. In contrast, a dominant error-prone route stemming from GMP misincorporation was observed for the major-groove O⁴-EtdT lesion. For the N3-EtdT lesion that disrupts base pairing, multiple transcriptional lesion bypass routes were found. Importantly, the results from the present in vitro transcriptional studies are well correlated with in vivo transcriptional mutagenesis analysis. Finally, we identified a minor-groove-sensing motif from pol II (termed Pro-Gate loop). The Pro-Gate loop faces toward the minor groove of RNA:DNA hybrid and is involved in modulating the translocation of minor-groove alkylated DNA template after nucleotide incorporation opposite the lesion. Taken together, this work provides important mechanistic insights into transcriptional stalling, lesion bypass, and mutagenesis of alkylated DNA lesions.

Keywords: DNA alkylation; RNA polymerase II; transcription; transcriptional lesion bypass; transcriptional mutagenesis.

Fig. 1.

RNA pol II transcriptional elongation in the damaged template containing an alkylated thymine base. (A) A schematic diagram showing the transcription elongation process, and the three steps of transcription checkpoint control, that is, insertion, extension, and proofreading. (B) Alkylation of thymine at different positions. (C) Scaffolds used in transcription elongation experiments. The position of damaged thymine base is marked as X. (D and E) Gel analysis of RNA pol II transcriptional elongation in the absence (D) and presence (E) of TFIIS. The concentration of NTP was 1 mM; the time points were 15 s, 1 min, 5 min, 20 min, and 1 h, respectively. The concentration of TFIIS is 1 µM.

Fig. S1.

ESI-MS and MS/MS characterizations of d(AGACGXTCCTCTCGATG), X = O²-EtdT. (A) Negative-ion ESI-MS; (B) the product-ion spectrum of the [M-5H]⁵⁻ ion (m/z 1,036.7).

Fig. S2.

ESI-MS and MS/MS characterizations of d(AGACGXTCCTCTCGATG), X = N3-EtdT. (A) Negative-ion ESI-MS; (B) the product-ion spectrum of the [M-5H]⁵⁻ ion (m/z 1,036.6).

Fig. S3.

ESI-MS and MS/MS characterizations of d(AGACGXTCCTCTCGATG), X = O⁴-EtdT. (A) Negative-ion ESI-MS; (B) the product-ion spectrum of the [M-5H]⁵⁻ ion (m/z 1,036.6).

Fig. S4.

RNA pol II transcriptional elongation in the presence of TFIIS. The concentration of TFIIS is 200 and 400 nM, respectively. The time points were 15 s, 1 min, 5 min, 20 min, and 1 h, respectively.

Fig. 2.

Nucleotide incorporation opposite the EtdT lesions. (A) A scheme illustrating the first fidelity checkpoint step (insertion) and the scaffold used in this assay. (B) Representative images of gels for monitoring single-nucleotide addition opposite the three ethylated thymine nucleosides. The concentration of NTP was 1 mM; the time points were 1 min, 5 min, 20 min, 1 h, 3 h, 8 h, and 1 d, respectively. (C) Kinetic analysis of single-nucleotide incorporation opposite the ethylated thymidines in comparison with the undamaged thymidine.

Fig. S5.

Kinetic analysis of k_pol for the incorporation and extension steps for the three regioisomeric EtdT lesions.

Fig. S6.

Representative kinetic fitting curves for nucleotide incorporation opposite to the N3-EtdT lesion (Upper) and extension past the N3-EtdT lesion (Lower).

Fig. 3.

Analysis of the subsequent extension step after the nucleotide addition opposite the alkylated thymine site. (A) Scaffold used in this assay. (B) Kinetic analysis of the subsequent extension after the ethylated thymine in comparison with the undamaged thymine base.

Fig. 4.

Backtrack and proofreading of RNA pol II after nucleotide insertion opposite the ethylated thymidine lesions. (A) Scaffold used in the cleavage assay. (B) Results of RNA pol II intrinsic cleavage. The black arrow refers to the pretranslocation cleavage product; the red arrow refers to the backtrack cleavage product. The time points were 5 min, 20 min, 60 min, 3 h, 8 h, 24 h, and 48 h, respectively.

Fig. S7.

TFIIS-stimulated RNA pol II backtrack and transcript cleavage. Time points were 10 s, 30 s, 1 min, 2 min, 4 min, 8 min, 15 min, 30 min, and 60 min, respectively.

Fig. 5.

Alkylation at different positions at the hydrogen bonding face of thymidine leads to distinct bypass preferences by RNA pol II. (A) Lesion bypass was controlled by different fidelity checkpoints. (B) A summary of transcription lesion bypass for the three regioisomeric EtdT lesions. Green, orange, and red arrows designate ribonucleotide incorporations that are efficient, difficult, and extremely difficult, respectively. This scheme was depicted based on the relative kinetic values of each transcriptional bypass step. (C) A comprehensive analysis of relative bypass efficiency of RNA pol II for the three regioisomeric EtdT lesions. (D) The distributions of nucleotides inserted opposite the regioisomeric EtdT lesions based on transcription assays conducted in XPA-deficient human skin fibroblasts, where the data represent the mean and SE of results from three independent transfection experiments (the panel was plotted based on data reported in ref. 41).

Fig. 6.

Transcriptional bypass of O⁴- and O²-EtdT has distinct structural effects. (A) During the incorporation of GMP, the guanine base can pair with both O⁴- and O²-EtdT. (B) In the extension step, the alkylation in the major groove (O⁴-EtdT) has limited disruption during translocation, whereas the minor-groove alkylation (O²-EtdT) has strong steric clash with the P448 residue, and further altered this minor-groove–interacting loop in Rpb1.

Fig. S8.

Energy minimization to minimize the steric clash between Rpb1 P448 from the Pro-Gate loop and O²-EtdT lesion. The color code is the same as Fig. 6. The energy-minimized Pro-Gate loop, O²-EtdT lesion, and GTP are highlighted in cyan, yellow, and orange, respectively.