Diversity of the damage recognition step in the global genomic nucleotide excision repair in vitro

The XPC-HR23B complex, a mammalian factor specifically involved in global g enomic nucleotide excision repair (NER) has been shown to bind various form s of damaged DNA and initiate DNA repair in cell-free reactions. To charact erize the binding specificity of this factor in more detail, a method based on immunoprecipitation was developed to assess the relative affinity of XP C-HR23B for defined lesions on DNA, Here we show that XPC-HR23B preferentia lly binds to UV-induced (6-4) photoproducts (6-4PPs) as well as to choleste rol, but not to the cyclobutane pyrimidine dimer (CPD), 8-oxoguanine (8-oxo -G), O-6-methylguanine (O-6-Me-G), or a single mismatch. Human whole cell e xtracts could efficiently excise 6-4PPs and cholesterol in an XPC-HR23B-dep endent manner, but not 8-oxo-G, O-6-Me-G or mismatches. Thus, there was goo d correlation between the binding specificity of XPC-HR23B for certain type s of lesion and the ability of human cell extracts to excise these lesions, supporting the model that XPC-HR23B initiates global genomic NER. Although , XPC-HR23B does not preferentially bind to CPDs, the excision of CPDs in h uman whole cell extracts was found to be absolutely dependent on XPC-HR23B, in agreement with the in vivo observation that CPDs are not removed from t he global genome in XP-C mutant cells. These results suggest that, in addit ion to the excision repair pathway initiated by XPC-HR23B, there exists ano ther sub-pathway for the global genomic NER that still requires XPC-HR23B b ut is not initiated by XPC-HR23B, Possible mechanisms will be discussed, (C ) 2001 Elsevier Science B.V. All rights reserved.