Cytosine analogues as DNA methyltransferase substrates

Area of Science:

• Biochemistry
• Molecular Biology
• Medicinal Chemistry

Background:

• DNA methyltransferases (DNMTs) are crucial epigenetic regulators and therapeutic targets for various cancers, including myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML).
• Understanding how nucleoside analogues interact with DNMTs is vital for developing effective anti-cancer drugs.

Purpose of the Study:

• To characterize the interaction of various nucleoside analogues with a model prokaryotic DNA methyltransferase (M.MpeI) to understand mechanisms relevant to mammalian DNMT1.
• To investigate the impact of different 5-substituents on DNA methyltransferase activity and complex formation.

Main Methods:

• Utilized a prokaryotic CpG-specific DNA methyltransferase (M.MpeI) as a model system for mammalian DNMT1.
• Tested nucleoside analogues including 5-hydroxymethylcytosine, 5-hydroxycytosine, 5-methylzebularine, 5,6-dihydro-5-azacytosine, 5-fluorocytosine, 5-chlorocytosine, 5-bromocytosine, and 5-iodocytosine.
• Employed mass spectrometry to elucidate reaction mechanisms and determined crystal structures of M.MpeI-DNA complexes.

Main Results:

• 5-fluorocytosine (5FC) demonstrated the highest efficiency in forming covalent complexes with M.MpeI.
• Non-covalent complexes were most abundant with 5,6-dihydro-5-azacytosine (dhaC) and 5-methylzebularine (5mZ).
• Surprisingly, 5-iodocytosine (5IC) and 5-bromocytosine (5BrC), and to a lesser extent 5-chlorocytosine (5ClC) and 5FC, underwent methylation via a methyltransferase-driven dehalogenation pathway in the presence of thiol nucleophiles.

Conclusions:

• The study provides insights into the interaction of modified nucleosides with DNA methyltransferases, highlighting 5FC as a potent covalent complex former.
• Methyltransferase-driven dehalogenation followed by methylation represents a novel reaction pathway for certain halogenated nucleoside analogues.
• Crystal structures reveal distinct active site loop conformations ('in' vs. 'out') dependent on the 5-substituent's steric bulk, with implications for DNMT1 and other DNA methyltransferases.