Positions of the repaired product, and two molecular weight markers are indicated by arrows.The ligation intermediate is a product resulting from addition of AMP to the terminus of the incised fragment by DNA ligase.B, structures of repair products, intermediates, and molecular weight markers.Excision of AP sites is a prerequisite to completion of base excision repair.Typically, it proceeds via an incision of the phosphate backbone at the side of the AP site followed by another incision at its side.Whereas the incision is mostly carried out through hydrolysis by AP endonuclease, the incision of AP sites can proceed via either belimination or hydrolysis.In higher eukaryotes, polbhas an activity for excision of a dRP residue from incised AP site through belimination. Thus, the pol bdependent pathway is able to complete repair of natural AP sites without additional buy Citalopram HBr factors for incision.On the other hand, FEN is able to excise a dRP residue from the incised AP site via hydrolysis. In this reaction, the terminal dRP residue is released together with its adjacent nucleotide. The hydrolytic excision by FEN can be applied not only to unmodified natural AP sites but also to modified or synthetic AP sites that are refractory to belimination.These characteristics of FEN confer two significant properties on the PCNAFIG.A, titration of ethidium bromide.The indicated concentrations of ethidium bromide were added to the repair reaction of either the or labeled AP sitecontaining DNA that had been predigested with AP endonuclease.B, stimulation of the excision activity of XFEN by PCNA and BE.When XFEN was added to this repair system, however, the synthetic AP site was efficiently repaired. Unlike PCNA and BE, which together stimulated excision by XFEN without DNA synthesis. B, stimulation of the excision activity of XFEN by polbor PCNA and BE.First, this pathway can repair modified or synthetic AP sites as efficiently as unmodified natural AP sites.Second, this pathway replaces at least two nucleotides during the repair reaction.These properties are distinct from those of the pol bdependent pathway, which repairs preferentially natural AP sites through the replacement of a single nucleotide. FEN can physically bind to PCNA with a stoichiometry of three FEN molecules to one PCNA trimer. The result of our gel mobility shift of PCNA with XFEN provides additional evidence for their strong interaction.They also observed a similar stimulation of FEN exonuclease activity by PCNA on circular DNA but not on oligonucleotide substrates.The stimulation of FEN by PCNA on circular DNA was detected with a reasonable molar ratio for their stoichiometric interaction.The simplest model provided from our result and the established function of PCNA and RFC is that FEN bound to PCNA is recruited to the nicked site by RFC.Because we employed the BE fraction instead of the purified RFC, it is still possible that other factors in BE, such as DNA polymerasedor DNA ligase, may also participate in the stimulation of XFEN.This result appears to contradict the result described here.One possible explanation to resolve this contradiction is that DNA polymerased may block the nuclease activity of FEN in the absence of DNA synthesis in the S extract, whereas an excess amount of FEN in the reconstituted system may overcome the blocking by DNA polymerase.