Due to its low redox potential, guanine (G) is the most frequent site of oxidation in the genome. Metabolic processes generate reactive oxygen species (ROS) that can oxidize G to yield 8oxo-7,8-dihydroguanine (OG) as a key two-electron oxidation product. In a genome, G-rich sites including many gene promoters are sensitive to oxidative modification, and some of these regions have the propensity to form G-quadruplexes (G4s). Recently, OG formation in G-rich gene promoters was demonstrated to regulate mRNA expression via the base excision repair (BER) pathway. The proliferating cell nuclear antigen (PCNA) gene was previously found to be activated by metabolic ROS, and the gene has a five G-track potential G4 in the coding strand of its promoter. Herein, we demonstrated the ability for four G runs of the PCNA promoter sequence to adopt a parallel-stranded G4. Next, we identified G nucleotides in the PCNA G4 sequence sensitive to oxidative modification. The G oxidation product OG and its initial BER product an abasic site were synthetically incorporated into the four-and five-track PCNA sequences at the sensitive sites followed by interrogation of G4 folding by five methods. We found the modifications impacted the G4 folds with positional dependency. Additionally, the fifth G track maintained the stability of the modified G4s by extrusion of the oxidatively modified G run. Finally, we synthetically inserted a portion of the promoter into a reporter plasmid with OG at select oxidation prone positions to monitor expression in human glioblastoma cells. Our results demonstrate that OG formation in the context of the PCNA G4 can lead to increased gene expression consistent with the previous studies identifying metabolic ROS activates transcription of the gene. This study provides another example of a G4 with the potential to serve as a regulatory agent for gene expression upon G oxidation.

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The discovery that bifunctional alkylating agents have antitumor activity led to the development of cancer chemotherapy over 50 years ago, yet these compounds also lead to an increased risk of secondary cancers. Bifunctional alkylating agents form a variety of DNA adducts, including monoadducts, intrastrand cross‐links, and interstrand cross‐links. Interstrand cross‐links are believed to be the most lethal, impeding both replication and expression of the genetic material. We are characterizing the mechanisms by which diepoxybutane (DEB) and epichlorohydrin (ECH) exert their cytotoxic effects in cultured cells. Our first goal is to determine the relationship between interstrand cross‐linking and cytotoxicity in human acute myeloid leukemia (HL60) cells. HL60 cells are treated with varying concentrations of the compound of interest and assayed to determine the fraction of cells that remain viable, allowing determination of LD50 values. Cross‐linking ability is assessed using an ethidium bromide assay to determine the amount of duplex DNA following alkaline denaturation. Only cross‐linked DNA reanneals rapidly and interacts with the dye, which is highly fluorescent when bound to duplex DNA. Preliminary results suggest a correlation between cross‐linking and LD50 values for these compounds, with DEB a more efficient cross‐linker than ECH.Our second goal is to identify the structures of ECH‐cross‐linked lesions via ESI‐TOF‐MS, which may provide information about the mechanism of this agent. This research was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423.