We have developed an affinity purification protocol for the isolation of uracil-DNA glycosylases (UDG) from human cells. This technique employs the uracil-DNA glycoyslase inhibitor protein derived from the PBS2 bacteriophage of B. subtilis. Using this approach, uracil-DNA glycosylase was purified from HeLa cells and analyzed by SDS-PAGE. After silver staining, a single 30 kDa band and a second group of proteins within the 36-38 kDa range are apparent. The bands within the 36-38 kDa range were transferred to PVDF membrane and each band subsequently digested with cyanogen bromide. Protein sequence, derived from peptides of three 36-38 kDa bands, was identical and revealed a unique terminal peptide that is not predicted by any previously identified UDG sequences. Synthetic primers were then generated to isolate the corresponding cDNA using PCR. The cDNA (UDGla) encodes 44 unique amino acids at its amino terminus yet retains the signals necessary for mitochondrial uptake and processing. In addition, the differences in migration on SDS-PAGE gels suggest that this protein may undergo post- translatjonal modifications. Transcription and translation of this cDNA m vitro yields a 37 kDa protein product that possesses uracilDNA glycosylase activity. Preliminary kinetic analysis of the in vitro translation products indicates that the specific activity of UDGla is significantly lower than that derived from the highly conserved UDG (both the precursor and processed forms). We are currently investigating the cellular location and 'in vivo role for UDG1a.
|Original language||English (US)|
|State||Published - Dec 1 1996|
All Science Journal Classification (ASJC) codes
- Molecular Biology