Oxidation of 5-methylcytosine: DNA damage and epigenetic reprogramming

The DNA of all organisms undergoes persistent damage that can result in genetic mutations or epigenetic changes unless the damage is correctly repaired. The modified base, 5-methylcytosine (5mC), can undergo both deamination and oxidation resulting in both genetic mutations and epigenetic perturbation. We have demonstrated that the oxidation damage product, 5-hydroxymethylcytosine (5hmC), inhibits the binding of proteins that selectively bind to methylated DNA and that the maintenance methyltransferase DNMT1 does not recognize 5hmC when methylating DNA following DNA replication. Therefore, the formation of 5hmC could heritably alter epigenetic patterns in replicating cells. Emerging evidence indicates, however, that the conversion of 5mC to 5hmC could also be part of an enzymatic epigenetic reprogramming pathway essential for stem cell differentiation and that 5hmC is absent from most if not all human cancer cells. 

We wish to understand how DNA demethylation relies upon DNA damage repair pathways, and how DNA damage repair and DNA demethylation might become entangled. We use novel stable isotope labeling method that will allow us to follow the dynamic processes of DNA replication, methylation and hydroxylation and excision of possible intermediates by the base excision repair pathway.

In addition we have develop a series of innovative new methods to identify and quantify enzymatic activities that might act on pathway intermediates, further defining as yet unknown parts of this pathway. The information obtained is essential for understanding the biology of normal stem cells within the context of regenerative medicine, and understanding how the pathway becomes defective in human cancer cells. Understanding these potential defects may provide new insights into novel targeted chemotherapy.