The Efficiencies of Damage Recognition and Excision Correlate with Duplex Destabilization Induced by Acetylaminofluorene Adducts in Human Nucleotide Excision Repair

Abstract

Nucleotide excision repair (NER) removes lesions caused by environmental mutagens or UV light from DNA. A hallmark of NER is the extraordinarily wide substrate specificity, raising the question of how one set of proteins is able to recognize structurally diverse lesions. Two key features of good NER substrates are that they are bulky and thermodynamically destabilize DNA duplexes. To understand what the limiting step in damage recognition in NER is, we set out to test the hypothesis that there is a correlation of the degree of thermodynamic destabilization induced by a lesion, binding affinity to the damage recognition protein XPC-RAD23B and overall NER efficiency. We chose to use acetylaminofluorene (AAF) and aminofluorene (AF) adducts at the C8 position of guanine in different positions within the NarI (GGCGCC) sequence, as it is known that the structures of the duplexes depend on the position of the lesion in this context. We found that the efficiency of NER and the binding affinity of the damage recognition factor XPC-RAD23B correlated with the thermodynamic destabilization induced by the lesion. Our study is the first systematic analysis correlating these three parameters and supports the idea that initial damage recognition by XPC-RAD23B is a key rate-limiting step in NER.

The relationships between XPC binding to conformationally diverse DNA adducts and their excision by the human NER system: Is there a correlation?

“…The lower band is attributed to a single XPC molecule-DNA complex and the higher one is attributed to complexes with two XPC molecules bound per DNA molecule. Similar double-molecule XPC-RAD23B -DNA complexes have been reported by other workers [10, 40–42]. However, when an adduct is present, the two bands are no longer distinguishable.…”

“…The NER excision efficiencies of adducts have been found to be correlated with the binding affinities of XPC-RAD23B [10]. …”

“…However, neither DDB2-DDB1 nor centrin 2 are required for the recognition of DNA adducts in cell extracts in vitro . The binding of the XPC-RAD23B heterodimer to the damaged DNA is the initial event [9, 10] that triggers the ensuing stepwise cascade of NER steps [11–15]. In order for the full processing of the DNA adducts to occur, it is believed that a two-step (bipartite) mechanism is required: (1) recognition by XPC-RAD23B and (2) the subsequent verification step that involves the helicase activity of XPD in TFIIH, the next multi-protein NER factor that binds to the XPC-RAD23B -DNA complex.…”

Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process

“…Because of the lack of direct contact with the lesion by Rad4, Rad4 (XPC) is proposed to indirectly recognize locally destabilized duplex DNA by probing the two strands’ propensity to open, which allows insertion of β-hairpin 3 (Min and Pavletich, 2007). This hypothesis provides a working model for how Rad4 (XPC) recognizes chemically and structurally diverse DNA damage in vitro , such as a cholesterol-modified nucleotide, 6-4PP, cisplatin 1,3-d(GTG) intrastrand adduct, C8-dG acetylaminofluorene, and 5R-thymine glycol (Brown et al, 2010; Hey et al, 2002; Jansen et al, 1998; Kusumoto et al, 2001; Sugasawa et al, 2002; Yeo et al, 2012). Previous studies on domain deletions and mutated XPC constructs employing bulk biochemical binding assays and a fluorescence-based cellular method suggest a two-stage damage recognition model.…”

Nucleotide Excision Repair in Eukaryotes

“…These properties were originally framed in the context of the bipartite recognition model that posits that good NER substrates are destabilizing and bulky (Hess et al 1997). Subsequent studies correlating the structures of adducts such as those formed by benzo[a]-pyrene or acetylaminofluorene with their repair efficiencies confirmed these observations and showed that factors such as disrupted base-pairing, bending, and flexibility can contribute to repair outcomes Cai et al 2010;Mu et al 2012;Yeo et al 2012). As discussed in the next section, we now understand these principles in terms of how damage recognition is achieved in NER.…”