Research Projects

The N7 atom of guanine is highly reactive toward electrophilic methylating agents. N7-methyl-Guanine (7meG) modification can arise from both endogenous metabolic processes and exogenous environmental exposures. S-adenosylmethionine (SAM), a cellular methyl donor, can cause spontaneous methylation at N7 of guanine, while methyl methanesulfonate (MMS), an exogenous alkylating agent, can be used to cause similar damage experimentally. If not repaired, 7meG lesions can compromise various cellular functions. The AlkB family of Fe(II)/α-ketoglutarate-dependent dioxygenases plays a critical role in reversing such damage via oxidative demethylation. The human genome encodes 8 ALKBH homologues (ALKBH1-8). While some AlkB homologs (ALKBH2, 3, 5) are well-characterised, the physiological roles of ALKBH1 and ALKBH6 remain largely unexplored. This project investigates the roles of these two enzymes in regulating 7meG lesions in nucleotides, DNA, and RNA (figure 1). Preliminary data from our lab demonstrate that ALKBH6 catalyses the demethylation of 7meG-containing nucleotides, particularly 7meGMP and 7meGDP. These 7meG nucleotides resemble the mRNA 5′ cap structure and may disrupt key cellular processes if not efficiently cleared. Their accumulation is hypothesized to: (a) compete with capped mRNAs for eIF4E binding, impairing translation initiation; (b) inhibit the decapping scavenger enzyme DcpS, thereby affecting mRNA turnover; and (c) mimic GDP and interfere with Ras GTPase signalling pathways that are crucial for protein synthesis, cell growth and survival. The inhibitory effects of 7meG nucleotides are expected to be more pronounced in cancer cells due to their higher need for protein synthesis. Another set of unpublished data from our lab suggests that ALKBH1 demethylates 7meG lesions from RNA and single-stranded DNA (ssDNA). While 7meG in double-stranded DNA is repaired via base excision repair (BER), the mechanism of 7meG removal from ssDNA and RNA remains unclear. These lesions, particularly in G-rich sequences, may disrupt Hoogsteen base pairing and impair the formation of non-canonical DNA structures such as triplex DNA and G-quadruplexes. Internal 7meG modifications are also introduced into mRNA and tRNA through the activity of the METTL1-WDR4 methyltransferase complex. Whether ALKBH1 demethylation activity contributes to reversing these 7meG methylation is a key question that will be investigated in this study. The overall goal of the project is structured around understanding the regulation of 7meG at the nucleotide and the nucleic acid level. One of the objectives is to find the link between ALKBH6 activity and the regulation of 7meG nucleotides and determine its impact on translation initiation. Further, efforts will be made to define the role of ALKBH1 in demethylating 7meG lesions from RNA and ssDNA. The project will also investigate the effects of 7meG incorporation in G-rich DNA motifs by evaluating triplex and G-quadruplex stability. To address these aims, a combination of experimental techniques will be employed. Biochemical assays will utilise synthetic 7meG-modified nucleotide and oligonucleotide substrates and recombinant ALKBH6 and ALKBH1 proteins, cellular assays will involve gene knockdown, reporter gene systems, immunofluorescence microscopy and flow cytometry. Collectively, these efforts aim to define the mechanistic roles of ALKBH6 and ALKBH1 in maintaining the quality of the nucleotide pool and nucleic acid structures by preventing the accumulation of 7meG methylated bases. This work has the potential to expand our understanding of RNA and DNA demethylation pathways and their broader implications in translation control, genome integrity, and stress responses. The project also seeks to develop 7meG-responsive synthetic DNA switches as molecular tools to monitor demethylation activity and cellular methylation stress.