A convenient spectrometric assay system for intracellular quantitative measurement of DNA glycosylase activity.
Cytosine methylation is a vital biology event. However, it is also the source of genomic instability due to deamination of 5'-methylcytosine by spontaneous hydrolysis, which produces thymine and results in G:T mismatches. Thymine DNA glycosylase and methyl-CpG-binding protein 4 are major DNA glycosylases involved in the mismatch repair progress, and their activities have been measured in many related researches. In this study, we developed a convenient spectrometric assay system for specific and quantitative measurement of intracellular DNA glycosylase activity. A G:T mismatch was introduced into the upstream region of firefly luciferase-coding sequence in the pGL3-control plasmid. Only if the G:T mismatches were repaired to G:C, will luciferase be expressed in transfected cells. By measuring luciferase activity, which is simple and convenient, the intracellular DNA glycosylase activity can be determined.
Li
Shiying
S
Jiangsu Center of Hepatobiliary Diseases and School of Stomatology, Affiliated Stomatological Hospital, Nanjing University, China.
Huang
Qilai
Q
Wang
Lu
L
Lan
Yan
Y
Zhang
Xin
X
Yang
Bingya
B
Du
Pan
P
Hua
Zichun
Z
D000818:Animals; D020137:Base Pair Mismatch; D045744:Cell Line, Tumor; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000032:analysis*; D017930:Genes, Reporter; D005822:Genetic Vectors; D049409:Luciferases, Firefly / Q000378:metabolism; D049410:Luciferases, Renilla / Q000032:analysis; D051379:Mice; D010957:Plasmids; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013941:Thymine / Q000378:metabolism; D044502:Thymine DNA Glycosylase / Q000032:analysis*
D049410:Luciferases, Renilla; D049409:Luciferases, Firefly; D004706:Endodeoxyribonucleases; C492249:Mbd4 protein, mouse; D044502:Thymine DNA Glycosylase; D013941:Thymine