Compared with the molecular size marker corresponding to the product that lost only the terminal tetrahydrofuran residue, these short products appeared to result from excision of the tetrahydrofuran together with one or more adjacent nucleotides from the incised AP site.We could not detect an intermediate resulting from excision of only the sugar phosphate residue.This is consistent with the purchase Tazobactam acid previous report that FEN cannot remove the terminal dRP residue as a free form but only as a part of an oligonucleotide. To examine whether XFEN can bind to PCNA, we subjected mixtures of PCNA and XFEN to native polyacrylamide gel electrophoresis followed by immunoblotting.The difference in their mobilities seems to result from their isoelectric points. The percentage of repaired DNA in each reaction, which was calculated after scanning the gel with a phosphorimage analyzer, is shown in the bottom panel.Thus, all of the subsequent experiments were conducted with circular DNA as a repair assay substrate.Ethidium bromide is known to strongly inhibit the activity of most of DNA ligases.We tested whether this DNA intercalating agent could specifically block the ligation step during AP site repair.Among the four steps in AP site repair, the incision by AP endonuclease turned out to be strongly inhibited by ethidium bromide. When the DNA substrates had been treated with AP endonuclease prior to the addition of ethidium bromide, neither DNA synthesis nor dRP excision was significantly suppressed by the addition of mgml ethidium bromide. However, this concentration of ethidium bromide blocked ligation and, therefore, more than of the repair of tetrahydrofuran sites.Therefore, we examined the dRP excision by XFEN under conditions in which the ligation step was blocked by mgml ethidium bromide.Because the BE fraction also contains pol d, it is still possible that DNA synthesis might be involved in stimulating the dRP excision carried out by the flap endonuclease activity of XFEN.As a result, the AP site repair was almost completely hindered, whereas XFEN still efficiently excised the AP site residues in the presence of PCNA and the BE fraction. The products resulting from excision by FEN lost the AP site residue and at least one adjacent nucleotide. In the absence of either PCNA or the BE fraction, however, XFEN scarcely catalyzed the excision reaction. This result indicates that XFEN, along with PCNA and RFC, can efficiently excise a dRP residue even from the terminus that does not form a stable flap structure.Our recent observation also indicated that a mouse cell extract repaired the tetrahydrofuran site by the pol bdependent pathway, although not efficiently.In these repair reactions, FEN may play a role in the pol bdependent pathway to excise the tetrahydrofuran residue.Therefore, we tested whether XFEN can assist the pol bdependent pathway to repair the synthetic AP site analog.A, structure of the doublestranded oligonucleotide substrate used for excision assay.A tetrahydrofuran residue and a P label are designated by D and an asterisk, respectively.In the excision assay with the oligonucleotide substrate, however, neither the purified PCNA protein nor the BE fraction containing the RFC activity increased the efficiency of removal of the terminal tetrahydrofuran residues by XFEN.