The search for active demethylation processes led to the discovery of 5-hydroxymethyl-2'-deoxycytosine (5hmC) in mammalian DNA. This modified base is formed post-replicatively by oxidation of 5methyl-cytosine (5mC) in the presence of TET deoxygenases. Futher oxidation products of TET oxidations, 5-formyl-dC (5fC) and 5-carboxy-dC (5caC) are substrates for base excision repair and removed by thymine DNA glycosylase (TDG). However to date it is still unknown which enzyme is responsible for 5hmC removal from DNA. In this work we present studies of the modified Dickerson-Drew Dodecamer 5'-CGC GAA TTX GCG-3' (DDDOH), where X is 5mC, 5hmC, 5fC or 5caC. The high-resolution NMR results show that base pairing and base stacking is maintained throughout the duplex. The NMR as a function of temperature describes the effect induced by modified bases on the DNA stability. High-resolution proton exchange NMR spectroscopy has been used to characterize kinetics of base-pair opening in nucleic acid duplexes. The rates of exchange of imino protons with solvent protons were measured by magnetization transfer from water for each DNA duplex, as a function of the concentration of exchange catalyst. It was proposed that TDG uses an extrahelical base recognition mechanism, which is facilitated by the destabilized base pair possessing kinetically enhanced base pair opening rates. Thus, the dynamic properties of DNA containing 5mC, 5hmC, 5fC and 5caC are crucial to understand the role of repair enzyme in damage recognition. In work presented herein we try to explain similarities and differences among all adducts and discuss their potential as a substrates for base excision repair mechanisms. This research was supported by NIH grants ES-05509 and CA-55678.

Dynamics of the oxidation products of cytosine in DNA

246th National Meeting (2013)

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