Sensitive detection of the binding of E2F to its promoter by exonuclease III- and BssHII-protection PCR assays.
For the non-radioactive, sensitive detection of the binding of transcription factor E2F to its binding site (E2 promoter), exonuclease III (ExoIII)- and BssHII-protection PCR assays were established. The binding of glutathione S-transferase E2F-1 (GST-E2F-1) fusion protein to its promoter protected the promoter against ExoIII- and BssHII-digestion. For the BssHII-protection PCR assay, a BssHII restriction site was made in the E2 promoter sequence by changing one base-pair next to its sequence. To detect E2F binding in ExoIII- or BssHII-protection PCR assays, the use of 3.13 fmol (5.00 ng) or 2.33 fmol (4.62 ng) of DNA (containing E2 promoters) and 0.325 microg (3.70 pmol) or 0.175 microg (2.00 pmol) of GST-E2F-1 protein, respectively, were found to be sufficient.
Han
B
B
Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Japan.
Hamana
H
H
Shinozawa
T
T
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004262:DNA Restriction Enzymes / Q000737:chemistry; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004591:Electrophoresis, Polyacrylamide Gel; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D008954:Models, Biological; D016133:Polymerase Chain Reaction / Q000379:methods*; D011485:Protein Binding; D056465:Retinoblastoma-Binding Protein 1; D016914:Ribonuclease H / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D056465:Retinoblastoma-Binding Protein 1; D014157:Transcription Factors; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D004262:DNA Restriction Enzymes; C091139:ribonuclease HII; D016914:Ribonuclease H