The reactions were carried out at C for hand the excision products were separated on an denaturing polyacrylamide gel.B, sequential addition type excision assay with saturating and limiting concentrations of XPC.The left panels show only the parts of the autorads containing the excision products; the top panel shows the results of different orders of addition performed with nM XPC and the bottom panel shows the same experiment performed with nM XPC.We conclude, then, that under no conditions tested is DNA preincubated with XPC repaired faster than DNA preincubated with XPA or XPA RPA repair factors.Since the band shift assays with two factors reveal ternary complex formation with certain combinations we wished to carry out excision assays following such twofactor incubations in order to find out if there was correlation between the results obtained in gel shift experiments and excision kinetic assays, and hence if the ternary complexes detected by the band shift assays were on the pathway for assembly of the excision nuclease.The results of kinetic assays with the other two combinations shows that XPA XPC first <a href="http://www.molbioglobal.com/archives/330"></a>
reaction yields an excision rate comparable to the XPA first reaction. In contrast, the XPC RPA combination slows the rate of excision relative to the preincubation with RPA alone. Thus, the twofactor preincubation results are consistent with those of the band shift assays in that twofactor combinations which appear to form specific complexes with damaged DNA lead to a faster rate of repair relative to the twofactor combination which failed to produce a specific ternary complex.Formation of PIC requires XPC but, due to technical problems, we were unable to address the issue of whether XPC is actually a component of PIC.By using improved experimental conditions we are now able to answer this question.This indicates that PIC contains both XPA and XPC.In contrast, PIC and PIC formed with five and sixrepair factors, respectively, exhibited the slower mobility when the MBP tag is on XPA but not when it is on XPC, relative to complexes formed without tagged repair factors. . XPC is present in preincision complex but not in preincision complexes and. A, PIC, which is formed by XPA, RPA, XPC, and TFIIH, contains XPC.Note that the complexes formed with MBPXPA XPA and nontagged XPC. Even though all three proteins bind to damaged DNA preferentially, the selectivity achieved by any one of the three is insufficient to explain the high specificity of the excision nuclease for damaged DNA. B, excision assay performed with premixed six repair factors. The bottom panel shows quantitative analysis of data from the top panel and a duplicate experiment.are included in this figure as well for direct comparison of all twofactor incubation experiments.We wish to discuss the problem of damage recognition and the current models for high specificity complex formation, and propose a model which incorporates data from previous experiments and findings presented in this paper.While such a model is esthetically pleasing we consider it unlikely for the following reasons.First, most of the sixexcision repair factors are readily separated from one another under relatively mild purification conditions which makes the notion of a repairosome a semantic rather than a biochemical issue.