RECOGNITION OF DNA-ADDUCTS BY HUMAN NUCLEOTIDE EXCISION-REPAIR - EVIDENCE FOR A THERMODYNAMIC PROBING MECHANISM

The mechanism by which mammalian nucleotide excision repair (NER) dete cts a wide range of base lesions is poorly understood, Here, we tested the ability of human NER to recognize bulky modifications that either destabilize the DNA double helix (acetylaminofluorene (AAF) and benzo [a]pyrene diol-epoxide (BPDE) adducts, UV radiation products) or induc e opposite effects by stabilizing the double helix (8-methoxypsoralen (8-MOP), anthramycin, and CC-1065 adducts). We constructed plasmid DNA carrying a defined number of each of these adducts and determined the ir potential to sequester NER factors contained in a human cell-free e xtract. For that purpose, we measured the capacity of damaged plasmids to compete with excision repair of a site-directed NER substrate. Thi s novel approach showed differences of more than 3 orders of magnitude in the efficiency by which helix-destabilizing and helix-stabilizing adducts sequester NER factors. For example, AAF modifications were abl e to compete with the NER substrate similar to 1740 times more effecti vely than 8-MOP adducts, The sequestration potency decreased with the following order of adducts, AAF > UV greater than or equal to BPDE > 8 -MOP > anthramycin, CC-1065. A strong preference for helix-destabilizi ng lesions was confirmed by monitoring the formation of NER patches at site specific adducts with either AAF or CC-1065. This comparison bas ed on factor sequestration and repair synthesis indicates that human N ER is primarily targeted to sites at which the secondary structure of DNA is destabilized. Thus, an early step of DNA damage recognition inv olves thermodynamic probing of the duplex.