Effect of Nitric Oxide on DNA Repair Dioxygenase - Human AlkB Homolog 2

DNA methylation is an epigenetic process that affects gene silencing, genomic imprinting and X-chromosome inactivation. Aberrant methylation is associated with imprinting-related diseases, DNA mutations, and cancer. Aberrant DNA methylation activates certain repair mechanisms such as Direct Reversal (DRR). In mammalian cells, human AlkB homolog 2 (ABH2) is the major housekeeping enzyme in this pathway. Its function is to remove a methyl group from the 1st position on adenine and/or 3rd position on cytosine in double-stranded DNA. Alkylating antineoplastic agents can act via methylating DNA and therefore inhibition of DNA repair machinery, such as Fe(II)-dependent enzymes, like ABH2, might augment this treatment. For this reason iron chelators have emerged as potential anti cancer drugs for their ability to inhibit this class of DNA repair enzymes. Recently our group has shown that nitric oxide can sequester cellular iron in the form of dinitrosyliron complexes. Hence, we hypothesized that nitric oxide could inhibit ABH2 though interaction with iron leading to inhibition of methylated DNA repair. To test this, we developed a novel in vitro method of detection for ABH2 activity. This method consists of measuring ABH2 activity using methylated DNA followed by detection with quantitative real-time PCR (qRT-PCR) analysis. The major advantage of this method is the ability to avoid known obstacles associated with other detection methods, like radioactivity. Also it provides specific, robust, and accurate results. Using this technique we have shown that nitric oxide can effectively inhibit the demethylation activity of ABH2. These results indicate that nitric oxide or nitric oxide generating compounds might be an important adjuvant therapy to existing chemotherapeutic alkylating agents.