Evidence for three thymine DNA glycosylases in human cell extracts: substrate specificities of thymine DNA glycosylase activities
Sibghat-Ullah Lari, Yao-Zhong Xu, Rufus S. Day
Med Sci Monit 2005; 11(2): BR41-49
Background:Purified human thymine DNA glycosylase (TDG) recognizes a G: T mispair in a CpG sequence context more strongly than in any other, in addition to its inactivity toward 2-aminopurine: T or 2,6 diaminopurine: T pairs. We investigated the multiplicity of TDG to establish a better relationship between in vitro G: T mismatch incision and in vivo repair of a G: T to a G: C pair.Material/Methods:Cell-free extract was prepared from A1235-MR4 human glioma cells grown in tissue culture. Fractions containing TDG activities were separated on a strong anion-exchange column. 45-bp DNA containing a single G: T or altered G: T mispair was prepared for measuring mismatch-specific strand-incision.Results:The extract yielded three fractions containing TDG activities. Each was further purified on a sizing column to exclude a relationship between a small fragment and TDG activity. While the substrate activity range of fraction III, eluting at the highest salt concentration, was the same as the known TDG, fractions eluted at medium and low concentrations were distinct: fractions I and II reacted with substrates of known TDG and DNA containing or 2-aminopurine: T (2,6-diaminopurine: T) base pairs. Modified m4T mispaired with G in DNA was acted on by fraction I and not II or III, suggesting fraction I activity is distinct. Each fraction showed strong activity on DNA with G: U and G: T mispairs in the CpG sequence context.Conclusions:The unique range of each TDG activity corresponding to the three fractions indicates that human cells possibly express three distinct TDGs.
Keywords: Cell Extracts - chemistry, Thymine - metabolism, Thymine DNA Glycosylase - isolation & purification, Thymine DNA Glycosylase - metabolism, Base Pairing, Base Sequence, Cell Extracts - chemistry, Cell Line, Tumor, DNA - metabolism, Glioma, Guanine - metabolism, Humans, Molecular Sequence Data, Substrate Specificity, Thymine - metabolism, Thymine DNA Glycosylase - metabolism