The interaction of DNA polymerases with N3-methyladenine and related analogs

DNA damage plays an essential role in both the formation of cancer via carcinogens and the treatment of cancer via antineoplastic agents. When using antineoplastic agents for the treatment of cancer, the biological end points can lead to either a positive clinical outcome (killing the cancer cells) or a negative outcome (mutations leading to secondary cancers from the initial treatment). The cytotoxicity and mutagenicity is determined by the DNA damage generated and by how the DNA damage is processed by the cell. Most carcinogens and antineoplastic agents generate a diverse spectrum of DNA lesion. This complicates both the clinical outcomes of chemotherapeutic agents and the biological consequences of the damage generated.;We have generated DNA alkylating agents that leads to the formation of a specific form of DNA damage, N3-methyladenine (3-MeA). One of the methylating agents, Me-lex, has been shown to generate high levels of 3-MeA (∼90%). 3-MeA was shown to be highly cytotoxic and repaired by the base excision repair process. 3-MeA was shown to be weakly mutagenic and the mutation spectrum showed only a small set sites had a higher prevalence of mutations.;The interaction of DNA polymerases with DNA damage is an essential step in the eventual biological endpoint of the damage. Because of its inherent instability 3-MeA has been unable to be synthesized into DNA substrates suitable for DNA polymerase interactions. Using the minor-groove methylating agent (Me-lex), we have designed an assay that generates 3-MeA containing substrates suitable for their interaction with DNA polymerase. The ability of 3-MeA and stable structural analogs, 3-methy1-3-deazaadenine (3-MezA) and 3-deazaadenine(3-zA), to stall DNA polymerization was elucidated using DNA replication and insertion assays. Both 3-MeA and 3-MezA were strong blocks to DNA replication and insertion in all the polymerases tested. 3-zA was a weak block to replication in all the polymerases tested. The role that the local sequence of the site previously determined to be a site of mutagenesis on DNA replication was also explored.