Furthermore, it was reported that preincubation of damaged DNA with XPC protein followed by addition of other repair factors in the form of a partially purified cell extract resulted in a higher rate of repair relative to reaction conditions in which damaged DNA was incubated first with XPA or XPA RPA prior to addition of extract containing the additional repair factors. In addition, we used repair factors purified to homogeneity to conduct repair kinetic experiments under a variety of order of addition conditions.Of the three damage recognition proteins tested, we find the best discrimination between damaged and undamaged DNA with RPA protein followed by comparable levels of discrimination for the XPA and XPC proteins.Significantly, we find that preincubation of damaged DNA with XPA, RPA, or XPA RPA results in about fold faster rate of repair relative to DNA preincubated with XPC prior to addition of other repair factors.The sequence and preparation of this substrate have been described elsewhere. In supershift assays DNA and protein were preincubated, antibody was added to the reaction mixture, and following an additional min incubation, the samples were loaded onto the polyacrylamide gel.The reaction mixture was incubated at C for the indicated times and then DNA was extracted with phenol:chloroform and separated on denaturing polyacrylamide gels.Since the role of XPC in damage recognition is a critical question addressed in this study the amount of XPC necessary for optimal excision reaction was titrated carefully.In addition to the standard excision assay, we conducted omission type and <a href="http://inhibit09.online/archives/223"></a>
sequential addition type assays.In omission type assays, the substrate was incubated with all but one of the repair factors for min, then the omitted factor was added and further incubation continued at C.In sequential addition assays, the substrate was first incubated with one repair factor for min, and then the rest of the repair factors were added and incubation was continued at C.The reactions were stopped by quickfreezing on dry ice, and the excision products were analyzed by autoradiography following resolution in sequencing gels.A bp duplex photoproduct were incubated at C for min with the polypeptides at the indicated concentrations and the free fractions were separated on nondenaturing polyacrylamide gel.The quantitative analyses of the data are shown in the right panels.Bars indicate the standard error of two independent sets of experiments including the ones shown in this figure.To calculate the KS the measured binding constants with damaged DNA were adjusted for the frequency of the photoproduct. The KNS values agree with the previously published values for XPA within a factor of two.XPC is the protein with the highest affinity for both damaged and undamaged DNA and RPA has the best discriminatory power.The selectivity factor, which is defined as the ratio of equilibrium dissociation constant for the, is about for RPA, for XPA, and for XPC.We reasoned that the three subunits may act in a cooperative fashion to achieve the high specificity observed in vivo.However, with label in the complementary strand, a specific XPC effect was seen.Visual inspection of this figure as well as quantitative analysis of densitometric scans show no difference between the degree of protection of undamaged and damaged DNA by XPC protein.