Recognition of DNA Adducts by Human Nucleotide Excision Repair

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 helixstabilizing adducts sequester NER factors. For example, AAF modifications were able to compete with the NER substrate ϳ1740 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. Nucleotide excision repair (NER)1 is an essential pathway for removing bulky base modifications from DNA. This repair mechanism involves endonucleolytic cleavage at two phosphodiester bonds, one 3Ј and the other 5Ј of the site of damage, followed by excision of DNA damage as the component of a single-stranded fragment (1-5). The excised oligonucleotide is replaced by DNA repair synthesis, and DNA continuity is reestablished by ligation. In mammalian cells, the major sites of incision are at the 5th phosphodiester bond 3Ј and the 24th phosphodiester bond 5Ј to the lesion (6).

Dynamic two-stage mechanism of versatile DNA damage recognition by xeroderma pigmentosum group C protein

“…Instead, the observed substrate versatility of the GGR pathway implies that its promiscuous initiator, XPC protein, acts by recognizing damage-induced distortions of the DNA helix rather than specific base modifications. In support of this hypothesis, it has been observed that the GGR system exhibits a general preference for base adducts that lower the melting temperature of double-stranded DNA [45], suggesting that XPC protein may detect the single-stranded character of damaged sites carrying bulky lesions. However, not in all cases the degree of duplex destabilization correlates with excision efficiency.…”

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

“…Psoralen monoadducts and crosslinks are, at least in part, recognized and repaired by NER in human cells (39)(40)(41)(42)(43)(44)(45)(46). Psoralen monoadducts are somewhat helix-stabilizing, whereas crosslinks are helix-destabilizing (47,48). If XPA and RPA recognize helical distortions, then we would not expect a psoralen monoadduct to be recognized with the same efficiency as a psoralen crosslink.…”

Dynamics of a Benzo[a]pyrene-derived Guanine DNA Lesion in TGT and CGC Sequence Contexts: Enhanced Mobility in TGT Explains Conformational Heterogeneity, Flexible Bending, and Greater Susceptibility to Nucleotide Excision Repair

“…44 Since a variety of lesions are recognized, it has been suggested that the NER factors do not recognize the lesion itself, but the local distortions in the DNA that are associated with the lesions. 15,[44][45][46] In this connection, we have shown recently that the human NER factor XPC/HR23B is the first mammalian NER factor that recognizes anti- [BP]-N 2 -dG lesions; this factor distinguishes between the BD (+)-cis-, and MG (+)-trans-and (−)-trans-[BP]-N 2 -dG lesions by opening the duplex to different extents and at different sites in the vicinity of the lesions. 47 Gunz et al have proposed a thermodynamic probing mechanism based on their observations that differences of more than 3 orders of magnitude are observed in the efficiency by which helix-destabilizing and helix-stabilizing adducts are excised by the NER mechanism.…”

“…47 Gunz et al have proposed a thermodynamic probing mechanism based on their observations that differences of more than 3 orders of magnitude are observed in the efficiency by which helix-destabilizing and helix-stabilizing adducts are excised by the NER mechanism. 15 According to their bipartite model of NER, the NER machinery first recognizes disruptions of Watson-Crick hydrogen bonding, followed by an unspecified second verification step that ensures that a chemically modified nucleobase is indeed present. 7,8,16,48,49 This has been called an "indirect readout mechanism" by Dip et al 48 Our detailed structural analyses based on MD simulations have previously provided further insights into the nature of DNA distortions that provoke NER susceptibility.…”

“…Disturbed Watson-Crick pairing, impaired base stacking, and DNA bending are among the recognition hallmarks that have been proposed. [13][14][15][16] NMR solution studies of bulky carcinogen-DNA adducts have provided considerable insights into adduct conformations. 17,18 An intriguing finding is the importance of base sequence context in governing the conformations of such DNA adducts.…”