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.
Jiangsu Center of Hepatobiliary Diseases and School of Stomatology, Affiliated Stomatological Hospital, Nanjing University, China.
Shiying
S
Li
Qilai
Q
Huang
Lu
L
Wang
Yan
Y
Lan
Xin
X
Zhang
Bingya
B
Yang
Pan
P
Du
Zichun
Z
Hua
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