Anticancer drugs that alkylate DNA in the minor groove may give rise to 3-alkyl-adenosine adducts that interfere with replication, inducing apoptosis in rapidly dividing cancer cells. However, translesion DNA synthesis (TLS) by polymerase enzymes (Pols) with the capacity to bypass DNA adducts may contribute to damage tolerance and drug resistance. 3-Alkyl-adenosine adducts are unstable and depurinate, which is a barrier to addressing chemical and enzymatic aspects of how they impact the progress of DNA Pols. To characterize structure-based relationships of 3-adenine alkylation relevant to cancer drugs on duplex stability and DNA Pol-catalyzed DNA synthesis, we synthesized stable 3-deaza-3-alkyl-adenosine analogues, including 3-deaza-3-phenethyl-adenosine and 3-deaza-3-methoxynaphthylethyl-adenosine, and incorporated them into oligonucleotides. A moderate reduction of duplex stability was observed on the basis of thermal denaturation data. Replication studies using purified Y-family human DNA Pols hPol η, κ, and ι indicated that these enzymes can perform TLS over the modified bases. hPol η had higher misincorporation rates when synthesizing opposite the modified bases compared with adenine, whereas hPol κ and ι maintained high fidelity. These results provide insight into how alterations in chemical structure reduce bypass of minor-groove adducts, and provide novel chemical probes for evaluating minor-groove DNA alkylation.

SignificanceThe impact of DNA lesions on replication and mutagenesis is of high relevance for human health; however, the role of lesion-induced transcriptional mutagenesis (TM) in disease development is unknown. Here, the impact of O6-methylguanine–induced TM on p53 function as a tumor suppressor was investigated in human cells. Results showed that TM in 15% of the transcripts resulted in a reduced ability of p53 protein to transactivate genes that regulate cell-cycle arrest and induction of apoptosis. This resulted in the loss of functional cell-cycle checkpoints and in impaired activation of apoptosis, both canonical p53 tumor-suppressor functions. This work provides evidence that TM can induce phenotypic changes in mammalian cells that have important implications for its role in tumorigenesis.

SignificanceRNA polymerase II (Pol II) catalyzes the transcription of DNA to RNA in the nucleus. DNA alkylating cancer drugs can stall transcription; however, the basis for Pol II stalling when encountering a DNA template with minor-groove alkylation adducts has remained elusive due to its inherent chemical instability. We characterized the behavior of Pol II in transcription over minor-groove alkylation adducts and uncovered a previously unobserved mode of Pol II stalling wherein clashes between DNA adducts and the mobile trigger loop of RNA Pol II prevent translocation of the enzyme after nucleotide insertion. These results provide a molecular basis for how DNA damage in transcribed portions of the genome initiates DNA repair contributing to drug resistance.

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Regulatory toxicology helps define the balance between health risk and societal benefit of chemicals by applying a science-based approach, thus representing a potential career opportunity for scientists involved in biomedical research.