| Summary: | 碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 106 === Uracil is a normal base in RNA. However, in DNA it can arise from spontaneous deamination of a deoxycytidine residue. Reactive oxygen species from normal aerobic respiration as well as exposure DNA to nitrate can also enhance the formation of uracil. Uracil in DNA is potentially mutagenic since it prefer to pair with dATP during replication, yielding G-C to A-T transition mutation. Uracil DNA glycosylase (UDG) acts as a key component in base excision repair (BER) pathway to repair hydrolytic deamination of cytosine in DNA, thus is very important in maintaining genome integrity.
The abnormal UDG activity in human cells may cause malfunction of uracil excision repair and eventually various diseases. In recent years, new methods have been published to conveniently detect UDG activity. Traditional methods involved in radioactive labeling coupled with gel-electrophoresis or chromatography. Alternative approaches such as fluorescence, electrochemical and G-quadruplex assay are also applicable. However, most of these methods required specific labeling or had limitation of specific sequence design, not suitable for standardization of UDG measurement.
Herein, we designed non-labeled and non-radio-isotopic and very specific method to measure UDG activity. An oligodeoxyribonucleotide with a single uracil is annealed to a template DNA forming a defined G-U mismatch and is hydrolyzed by Escherichia coli UDG. Resulting product containing an apurinic/apyrimidinic (AP) site is subjected to Matrix Assisted Laser Desorption/Ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. The cleavage of uracil is identified by the mass change from uracil substrate to AP product. The high resolution of MS results clearly separate product signal from substrate signal.
We evaluated the UDG kinetic assay by MALDI-TOF MS. Assay UDG activity in short time course could obtain the initial rate of the reaction. The value of Vmax and Km were 1.02 sec-1 and 51.3 nM, which were consistent with traditional assay. To evaluate the suitable enzyme concentration for subsequent experiments for substrate specificity, we titrated the UDG cleavage efficiency using 50 pmole of DNA substrate. We concluded that applying 0.05U UDG yielded the best result. We examined UDG activity with single strand and double strand DNAs containing single uracil at various positions of center, near 5’ end, or near 3’end. We found that UDG showed three fold active on single-stranded DNA substrate than that of double-stranded reactions. And we also found that UDG reacted more effectively when dU was at 5’ end of DNA substrate.
For a protocol safer and amenable to automation, we tested an alternative quenching protocol with HCl/DEA yielded similar results. We designed and tested endonuclease-resistant DNA substrate with chemical modification intend in vitro human cell assay. We also demonstrated the activity of hSMUG1 which is one of UDG in human cells by MALDI-TOF MS. We also subject uracil substrate to direct comparison of E. coli and mammalian UDG measurements, and we found inconsistency of units defined by different assays from the same commercial source of the enzymes. For the convenient, accurate, precision of this MALDI-TOF analysis should make it potentially a reference method in the future. Uracil DNA Glycosylase inhibitor (UGI) was employed to evaluate inhibition kinetic. The results should demonstrate the high potential for pharmaceutical application of glycosylase inhibitor screening.
|
|---|
