The next step in this mechanism is the incision of the phosphate backbone immediately to the AP site by AP endonuclease, generating a hydroxyl terminus and a terminus with a deoxyribose phosphate group.The dRP residue is then excised, and a DNA polymerase fills in the gap.Finally, the DNA strand is sealed by the action of a DNA ligase.The costs of publication of this article were defrayed in part by the payment of page charges.Consequently, the pol bdependent pathway can repair an unmodified natural AP site efficiently but not a reduced AP site or a synthetic AP site analog, hydroxyhydroxymethyltetrahydrofuran, neither of which is <a href="http://inhibit09.online/archives/210"></a>
susceptible to belimination.Although BEB and BE have not been purified as homogeneous proteins, the requirement of these fractions for DNA synthesis, excision of AP sites, and ligation steps suggests that replication factor C, a exonuclease, and a DNA ligase are included in one or the other of these fractions.Flap endonuclease was originally isolated as a DNA structurespecific endonuclease that cleaves a flap strand of branched DNA with a singlestranded terminus at the position near its junction to the doublestranded structure. Thus FEN carries several distinct nuclease activities on specific structured DNA substrates.Furthermore, recent studies demonstrate that PCNA directly binds to FEN and stimulates its activity. Positive phage clones were isolated and converted to plasmids according to the manufacturers instructions was chosen for further characterization.Each subclone was sequenced with a reverse primer and a T primer with an automated sequencer. These mutant proteins were overproduced and purified in the same manner as the wildtype FEN protein.Tween overnight.Among these substrates, the labeled DNA has a phosphate at the position nucleotides away from the lesion toward, whereas the labeled DNA has a P atom at the position nucleotides away from the lesion toward. Repair assay with these covalently closed circular DNA substrates was conducted as described previously. Briefly, the indicated proteins were incubated with either the or labeled DNA containing a tetrahydrofuran site at C for min.mg of protein of the BE fraction were employed.unit of T DNA ligase were used.The prelabeled oligonucleotide substrate was prepared by annealing the two oligonucleotides and endfilling at the end of the lesioncontaining strand with dATP and reverse transcriptase.mgml bovine serum albumin.After incubation at C for min, the reaction mixtures were mixed with ml of sequencing gel loading solution, boiled for min, and subjected to electrophoresis on a polyacrylamide gel containing M urea at V for h.To examine whether the BE fraction contains the XFEN protein, we prepared a polyclonal antibody against recombinant XFEN and used it for immunoblotting.Human FEN proteins carrying the corresponding mutations have been reported to lose flap endonuclease activity. The XFEN mutant proteins also lost the activity for supporting AP site repair, ruling out the possibility that contaminated bacterial proteins, rather than XFEN, might complement the repair activity.Taken together, these results are consistent with the hypothesis that XFEN is the essential factor for the activity of the BE fraction in AP site repair.In this experiment, we employed a doublestranded oligonucleotide substrate carrying a tetrahydrofuran residue in the middle of one strand.