Cooperative Interaction of Human XPA Stabilizes and Enhances Specific Binding of XPA to DNA Damage

Abstract

Human xeroderma pigmentosum group A (XPA) is an essential protein for nucleotide excision repair (NER). We have previously reported that XPA forms a homodimer in the absence of DNA. However, what oligomeric forms of XPA are involved in DNA damage recognition and how the interaction occurs in terms of biochemical understanding remain unclear. Using the homogeneous XPA protein purified from baculovirus-infected sf21 insect cells and the methods of gel mobility shift assays, gel filtration chromatography, and UV-cross-linking, we demonstrated that both monomeric and dimeric XPA bound to the DNA adduct of N-acetyl-2-aminofluorene (AAF), while showing little affinity for nondamaged DNA. The binding occurred in a sequential and protein concentration-dependent manner. At relatively low-protein concentrations, XPA formed a complex with DNA adduct as a monomer, while at the higher concentrations, an XPA dimer was involved in the specific binding. Results from fluorescence spectroscopic and competitive binding analyses indicated that the specific binding of XPA to the adduct was significantly facilitated and stabilized by the presence of the second XPA in a positive cooperative manner. This cooperative binding exhibited a Hill coefficient of 1.9 and the step binding constants of K 1 = 1.4 × 10 6 M -1 and K 2 ) = 1.8 × 10 7 M -1 . When interaction of XPA and RPA with DNA was studied, even though binding of RPA-XPA complex to adducted DNA was observed, the presence of RPA had little effect on the overall binding efficiency. Our results suggest that the dominant form for XPA to efficiently bind to DNA damage is the XPA dimer. We hypothesized that the concentration-dependent formation of different types of XPA-damaged DNA complex may play a role in cellular regulation of XPA activity.The human XPA (xeroderma pigmentosum group A) protein is an indispensable protein of human nucleotide excision repair (NER) that recognizes and removes a large variety of structurally distinct bulky DNA lesions (1-3). The XPA gene is required for both global genome repair (GGR) and transcription-coupled repair (TCR) (4,5). The protein with a zinc finger motif is capable of binding specifically to the damaged DNA in vitro. XPA interacts with the replication protein A (RPA) to form a protein-protein complex, and this complex formation is believed to be important for NER.A better understanding of the mechanism of human NER, and specifically determination of the role of human XPA, is being actively pursued by research groups including ours. Earlier reports suggest that XPA is involved in the DNA damage recognition process of NER and also † This study was supported by NCI Grant CA86927 (to Y.Z.) and NIEHS Grants ES09127 and ES 00318 (to A.K.B.)

Structural insights into the recognition of cisplatin and AAF-dG lesion by Rad14 (XPA)

“…S1). We confirmed complex formation with DNA containing a cisplatin 1,2-GG (10,36,41) and an AAF-dG lesion (37,38) (Fig. S1 D and E).…”

“…3 F and G and Fig. S1E) (37). In our model, the first Rad14 might bend the duplex slightly, thus facilitating the second Rad14 binding step, which provides the ability to form the sharp kink.…”

“…Although the function of most XP-proteins within the NER process is understood, the precise role of XPA has remained unclear XPA is known to interact with other NER proteins such as RPA, TFIIH, and ERCC1 (30)(31)(32)(33) suggesting XPA to be a NER scaffold protein (3). Furthermore, detailed biochemical experiments showed that XPA binds to kinked DNA structures (34) and to DNA duplexes containing certain types of DNA lesions, such as cisplatin (10,35,36) adducts and bulky adducts (5,37,38). XPA was found to form a homodimer (38), and it was demonstrated that it forms a 2:1 complex with the respective DNA (37).…”

“…Furthermore, detailed biochemical experiments showed that XPA binds to kinked DNA structures (34) and to DNA duplexes containing certain types of DNA lesions, such as cisplatin (10,35,36) adducts and bulky adducts (5,37,38). XPA was found to form a homodimer (38), and it was demonstrated that it forms a 2:1 complex with the respective DNA (37). Initial structural insights into the protein architecture were derived from an NMR structure of the DNA binding domain of XPA (39,40).…”

Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related–Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

“…In the mechanism of NER pathway, xeroderma pigmentosum group A (XPA) plays an indispensable role and is required for both global genome repair and transcription-coupled repair (6)(7)(8)(9). It is generally believed that XPA is involved in DNA damage recognition and also in the recruitment of other NER factors to the DNA damage site to form dual incision complexes through protein-protein interactions (8,(10)(11)(12).…”