title: Recognition of DNA adducts by human nucleotide excision repair. Evidence for a thermodynamic probing mechanism.
abstract: The mechanism by which mammalian nucleotide excision repair (NER) detects 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 induce 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 their potential to sequester NER factors contained in a human cell-free extract. For that purpose, we measured the capacity of damaged plasmids to compete with excision repair of a site-directed NER substrate. This 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 able to compete with the NER substrate approximately 1740 times more effectively than 8-MOP adducts. The sequestration potency decreased with the following order of adducts, AAF > UV >/= BPDE > 8-MOP > anthramycin, CC-1065. A strong preference for helix-destabilizing lesions was confirmed by monitoring the formation of NER patches at site-specific adducts with either AAF or CC-1065. This comparison based on factor sequestration and repair synthesis indicates that human NER is primarily targeted to sites at which the secondary structure of DNA is destabilized. Thus, an early step of DNA damage recognition involves thermodynamic probing of the duplex.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Zürich-Tierspital, Winterthurerstrasse 260, 8057 Zürich, Switzerland.
authors.forename: D
authors.initials: D
authors.lastname: Gunz
authors.forename: M T
authors.initials: MT
authors.lastname: Hess
authors.forename: H
authors.initials: H
authors.lastname: Naegeli
mesh_terms: D015073:2-Acetylaminofluorene; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000378:metabolism; D000876:Anthramycin; D000903:Antibiotics, Antineoplastic; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis* / Q000187:drug effects / Q000528:radiation effects; D018736:DNA Adducts / Q000032:analysis / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D000080890:Duocarmycins; D006367:HeLa Cells; D006801:Humans; D007211:Indoles; D007933:Leucomycins; D008730:Methoxsalen; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D015183:Restriction Mapping; D013379:Substrate Specificity; D013816:Thermodynamics; D014466:Ultraviolet Rays
chemical_list: D000903:Antibiotics, Antineoplastic; D018736:DNA Adducts; D000080890:Duocarmycins; D007211:Indoles; D007933:Leucomycins; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D000876:Anthramycin; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; C018782:CC 1065; D004247:DNA; D015073:2-Acetylaminofluorene; D008730:Methoxsalen