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An implication emerging from these observations is that with certain substrates or with all substrates in special structures as exist during transcription, XPC is not needed for excision nuclease activity.Consequently, XPFERCC may be the last factor to join the assembly.In contrast, no <a href="https://www.ncbi.nlm.nih.gov/pubmed/15820858"></a> incision was observed in   the absence of XPG XPG must be present to form the preincision complex; and the incision made by XPFERCC depends on the presence of the incision made by XPG, but not on the actual existence of XPG in the preincision complex.Previous studies have shown that inhibition of incision by antiXPG antibodies does not interfere with the incision, indicating that the formation of incision is not dependent on the production of the nick. Hence, the failure to observe incision in the absence of XPG might be due to the fact that XPG must be present in the preincision complex to enable XPFERCC to bind and carry out the incision.To test this model, we conducted the following experiment.CFE from an XPF or XPG mutant cell line was mixed with substrate that contained biotinylated nucleotides at the termini.Following incubation, the substrate and preincision complexes on the substrate were removed from the unbound cellular proteins using streptavidinattached magnetic beads.An XPG or XPF cellfree extract was then added to the immobilized complexes isolated from XPF or XPG CFE, respectively.In contrast, addition of XPF CFE to the complexes pulled down from the XPG extract in the same manner did not result in excision. Substrate was excised, and. B, excision of cholesterolB lesion.The positions of the main excision products are shown by a bracket.Cellfree extracts from XPF were mixed with internally radiolabeled cholesterolA substrate with a biotin tag.Lane, the XPF plus XPG cellfree extract was used as a positive control.Lane, the substrate pulled down in XPF CFE with streptavidin beads was incubated in XPG CFE.Lane, the substrate pulled down following incubation with XPG CFE by beads was then incubated with XPF CFE.However, in a previous study, it was found that antiXPG antibodies inhibited incision while having only a modest effect on incision, whereas antiERCC antibodies inhibited both and incisions to the same extent. These observations are consistent with incision preceding and being necessary for incision.To investigate the order of incision directly, a kinetic experiment was performed to examine the appearance of the two incisions.shows the results of this experiment, and the upper panel shows the quantitations of uncoupled nicks and excision that represents the nicks coupled to nicks on internally radiolabeled substrate.At early time points was detectable, suggesting that incision happens first.While the uncoupled nicks seemed to level off after min, dual incision continued to accumulate.Aliquots were taken at the indicated time points, quenched with g of proteinase K, and analyzed on an sequencing gel.The uncoupled incision appeared before the excision product, which required both and incisions.Thus, analogous to the bacterial excision nuclease, we conclude that in human excision nuclease, the incision precedes the incision.Its polypeptide composition ranges from to depending on the purification procedure. The precise function of TFIIH in excision nuclease is not known; however, two of its subunits, XPB and XPD, have helicase activities that are thought to be important for locally unwinding the duplex and thus targeting the nuclease subunits to their proper sites.

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