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

Abstract

The XPC–HR23B complex, a mammalian factor specifically involved in global genomic nucleotide excision repair (NER) has been shown to bind various forms of damaged DNA and initiate DNA repair in cell-free reactions. To characterize the binding specificity of this factor in more detail, a method based on immunoprecipitation was developed to assess the relative affinity of XPC–HR23B for defined lesions on DNA. Here we show that XPC–HR23B preferentially binds to UV-induced (6-4) photoproducts (6-4PPs) as well as to cholesterol, but not to the cyclobutane pyrimidine dimer (CPD), 8-oxoguanine (8-oxo-G), O⁶-methylguanine (O⁶-Me-G), or a single mismatch. Human whole cell extracts could efficiently excise 6-4PPs and cholesterol in an XPC–HR23B-dependent manner, but not 8-oxo-G, O⁶-Me-G or mismatches. Thus, there was good correlation between the binding specificity of XPC–HR23B for certain types 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 human 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 the global genome in XP-C mutant cells. These results suggest that, in addition to the excision repair pathway initiated by XPC–HR23B, there exists another sub-pathway for the global genomic NER that still requires XPC–HR23B but is not initiated by XPC–HR23B. Possible mechanisms will be discussed.