While a complex of moderate stability of a subset of excision nuclease subunits is still a possibility we believe that the preponderance of available data are against the existence of a suprarmolecular repairosome complex.Indeed, data exist which show that XPA and RPA make a complex with higher selectivity than either factor alone. Thus, while the concerted damage recognition model has not been rigorously eliminated, neither is there direct experimental support for such a model.High specificity binding is achieved by a cascade whereby the selectivities of XPA, RPA, and XPC are multiplied instead of being added up as in the concerted model.Based on repair kinetics under a variety of order of addition experiments, a previous study concluded that when damaged DNA was incubated with XPC first the reaction proceeded faster and therefore it was concluded that XPC is the first protein to bind to damaged DNA. In the present study we find the opposite: when the substrate is incubated with XPA, RPA, or XPA RPA first, the reaction proceeds at a faster rate than the reaction in which the DNA was first incubated with XPC.We do not have a satisfactory explanation for these contradictory results.However, it might be useful to point out the differences in the ways the two studies were performed.Whether these experimental differences explain the contradictory results remains to be determined.However, under no circumstances, including using cellfree extract for the repair assay, have we observed faster repair rates by incubating the substrate first with XPC and then supplementing with the other repair factors in the form of cellfree extract from an XPC mutant cell line. Thus, we conclude that if the assembly of human excision nuclease is by a sequential mechanism XPC cannot be the first factor to bind to the damage sites.First, RPA is an abundant cellular protein and, of the three excision repair proteins with preference for damaged DNA, it is the one with the highest selectivity factor.Second, RPA and XPA are required for recognition and removal of all DNA lesions regardless of the type of lesion or the DNA structure around or in the vicinity of the lesion. Third, in the defined excision nuclease system preincubation of DNA with RPA or RPA XPA leads to faster rates of repair relative to DNA preincubated with XPC.The resulting complex contains the four repair factors, and the DNA is actively unwound hence the name preincision complex. The following findings are consistent with PIC being the second step along the pathway of excision nuclease assembly.First, although XPC has the highest affinity for damaged DNA among all repair factors it has poor selectivity for damage and, as the kinetic experiments in this paper reveal, it must enter the complex after XPA and RPA for an optimum excision reaction.Second, damage recognition and excision occur in the absence of XPC under a variety of conditions: certain synthetic lesions, and thymine dimers blocking progression of RNA polymerase II are recognized and removed without XPC.Although under certain experimental conditions incision and in PIC formed with an active site mutant XPG incision can occur in the absence of incision, under optimal reaction conditions the dual incisions occur in a concerted manner.