Developing DNA probes to trap DNA cytosine-5 methyltransferases involved in promoter hypermethylation in cancer.

摘要

A major limitation to making significant advances in diagnosing and treating cancer is that we do not have a thorough understanding of the mechanisms leading to abnormal DNA methylation in cancer cells. Although there is a plethora of information on genes that are hypermethylated in cancer cells, we rarely know which DNA cytosine-5 methyltransferase (DNMTs) and what complex is involved in methylating particular promoters in cancer cells. I developed two types of DNA probes, a diazirine and a disulfide based, which can be incorporated at specific positions of oligonucleotides that have potential to trap DNMTs and their cofactors at a particular promoter. A diazirine photophore was introduced into either the major or minor groove of DNA via a convertible nucleoside methodology. The resulting DNA probes efficiently cross-linked with two different proteins studied as examples, the E. coli DNA adenine methyltransferase (EcoDam) and the human O6-alkylguanine-DNA alkyltransferase (hAGT). Efficient cross-linking of diazirine can be utilized to trap proteins from cell extracts. Taking advantage of DNMTs' invariant active site Cys and their base flipping property, a new disulfide-based DNA probe, 1'-methylenedisulfide deoxyribose, which can efficiently cross-link Haemophilus haemolyticus methylase (M. Hhal) and Spiroplasma sp. Methylase (M. SssI) was also developed. Compared to commercially available 5-FdC, the new probe cross-links DNMTs quickly. Using a disulfide tether on the N4 position of cytosine, catalytic domain of DNMT3A (DNMT3AC) has been cross-linked to DNA in high efficiency. Such a high cross-linking yield of DNMT3AC holds great promise in identifying DNMTs and their cofactors that act on particular promoters, as well as in structurally characterizing the DNMT3AC-DNA complex.