B, DNA was incubated with XPC monomer as indicated.In lanes and, antiXPA antibody was added to the binding mixtures before loading onto the gel.Under no conditions, however, were we able to obtain a specific footprint around the damage site.The footprinting experiments, nevertheless, revealed a specific effect of XPA and RPA on XPCDNA interactions never seen before in studies aimed at understanding the assembly of human excision nuclease.We wished to search for such a complex by conducting gel mobility shift assays.To better discern the cooperative interaction of the two repair factors, reactions were performed under conditions where band shift by the individual factors was minimal which can be supershifted by antiXPA antibodies and thus must contain XPA.Furthermore, the retarded band generated by XPA XPC monomer and hence the retarded bands in lanes and, in addition to XPA, must also contain the XPC protein either as a monomer or as a heterodimer.We used MBPXPC in these experiments so that we could conduct supershift experiments with commercially available antiMBP antibodies.Under the assay conditions used in this experiment both RPA give rise to discrete retarded bands.The combination of the two proteins yields the same two bands indicating the lack of a ternary complex containing both repair factors.B occurs by a relatively transient interaction between these two proteins or by an unknown mechanism.The mer was incubated either with the six premixed repair factors, or with five factors first followed by the addition of the omitted repair factor and samples were taken at the indicated time points.The top panels show the excision products detected by autoradiography following electrophoresis on denaturing polyacrylamide gels.The bottom panels show quantitative analyses of the data from the experiments in the top panel plus a second set of experiments performed under identical conditions.The substrate was incubated with repair factors in separate reactions missing XPA, XPC, or RPA; then the missing factor was added and incubation was continued.In all three instances the reaction rates were comparable.Each rate was significantly slower than the rate achieved when substrate was added to the mixture of all six repair factors.These results differ from those obtained with partially purified extracts where it was found that an XPA omission type of experiment yielded a repair rate at least fold faster than the XPC omission type of experiment. Clearly our results do not support the XPC first model.Hence we proceeded to test this model more directly by addition type assays.DNA was incubated with XPA, RPA, XPC, or XPA RPA for min, the remaining were added and incubation continued.Samples were taken for analysis at the indicated times.B, the top panels show the excision products detected by autoradiography following electrophoresis on denaturing polyacrylamide gels.Quantitative analyses of data from top panels and a second set of experiments conducted under identical conditions.In contrast, DNA preincubated with XPC was repaired about fold slower.These findings are not consistent with an XPC first model and actually suggest that, although XPC is capable of damage recognition, its binding to the lesion interferes with the proper assembly of the excision nuclease.

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