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 transcriptio n factor E2F to its binding site (E2 promoter), exonuclease III (ExoIII)- a nd BssHII-protection PCR assays were established. The binding of glutathion e S-transferase E2F-1 (GST-E2F-1) fusion protein to its promoter protected the promoter against ExoIII- and BssHII-digestion. For the BssHII-protectio n 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 E xoIII- 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 mu g (3.70 pm ol) or 0.175 mu g (2.00 pmol) of GST-E2F-1 protein, respectively, were foun d to be sufficient. (C) 1999 Elsevier Science B.V. All rights reserved.