National Research Council of Italy

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O6-alkylguanine-DNA alkyltransferase (AGT): lights on the scene

Rosanna Mattossovich 
IBBR - UOS Napoli - Italy

June 09, 2021 (11:30-12:30)
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Abstract: Cellular DNA is subjected to covalent modifications by intracellular chemical compounds and coming from the external environment. Alkylating agents are reactive molecules transferring chemical groups to nucleobases causing alterations in their functions. AGTs are enzymes which mainly remove alkyl adducts from the O6-position of guanines from DNA, by a peculiar irreversible reaction. Although their evolutive function represents the major factor in contrasting the effects of alkylating agents on DNA, on the other side the human representative of this class of enzymes (hMGMT) has a crucial clinical importance, because it contrasts the effect of chemotherapy’s based on alkylating agents, making tumor cells resistant. For these reasons. the development of hMGMT inactivators/inhibitors to be used in combination with this kind of chemotherapy is constantly evolving. As a consequence, rapid and reliable tests are needed for the measure of the methyltransferase activity. To this aim, DNA nanotechnology offers the possibility to create DNA nanodevices to monitor DNA repair activity. In this work, I present a new class of DNA-based substrates that, upon enzymatic DNA repair by AGTs, could undergo a conformational switch, followed by a change in a fluorescent signal. Such folding-uponrepair DNA single strand oligonucleotides, called DNA-nanoswitches, are synthetic DNA sequences containing as O6-methyl-guanine (O6-MeG) nucleobases, as well as a FRET fluorophores optical pair. These molecules are canonical DNA duplex, but they are rationally designed so that only upon enzymatic repair by demethylation of the O6-MeG nucleobases they can form stable intramolecular Hoogsteen interactions and fold into a DNA triplex structure, which is optically different from the initial DNA duplex form. I have characterized the folding mechanism induced by the enzymatic repair activity through fluorescent experiments and then I demonstrated that the folding-upon-repair DNA nanoswitches are universal AGTs’ substrates, successfully applying to several enzymes, including the hMGMT, the bacterial E. coli AdaC, and the archaea Saccharolobus solfataricus AGT. These innovative substrates will allow the high-throughput screening of alkylated DNA containing biological samples, as well as the selection of novel potential hMGMT inhibitors for cancer studies

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Istituto di Bioscienze e Biorisorse (IBBR/CNR)
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