Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair.

Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

Small Molecule Antagonists of the DNA Repair ERCC1/XPA Protein-Protein Interaction.

The DNA excision repair protein ERCC1 and the DNA damage sensor protein, XPA are highly overexpressed in patient samples of cisplatin-resistant solid tumors including lung, bladder, ovarian, and testicular cancer. The repair of cisplatin-DNA crosslinks is dependent upon nucleotide excision repair (NER) that is modulated by protein-protein binding interactions of ERCC1, the endonuclease, XPF, and XPA. Thus, inhibition of their function is a potential therapeutic strategy for the selective sensitization of tumors to DNA-damaging platinum-based cancer therapy. Here, we report on new small-molecule antagonists of the ERCC1/XPA protein-protein interaction (PPI) discovered using a high-throughput competitive fluorescence polarization binding assay. We discovered a unique structural class of thiopyridine-3-carbonitrile PPI antagonists that block a truncated XPA polypeptide from binding to ERCC1. Preliminary hit-to-lead studies from compound 1 reveal structure-activity relationships (SAR) and identify lead compound 27 o with an EC50 of 4.7 µM. Furthermore, chemical shift perturbation mapping by NMR confirms that 1 binds within the same site as the truncated XPA67-80 peptide. These novel ERCC1 antagonists are useful chemical biology tools for investigating DNA damage repair pathways and provide a good starting point for medicinal chemistry optimization as therapeutics for sensitizing tumors to DNA damaging agents and overcoming resistance to platinum-based chemotherapy.

The XPA Protein-Life under Precise Control.

Nucleotide excision repair (NER) is a central DNA repair pathway responsible for removing a wide variety of DNA-distorting lesions from the genome. The highly choreographed cascade of core NER reactions requires more than 30 polypeptides. The xeroderma pigmentosum group A (XPA) protein plays an essential role in the NER process. XPA interacts with almost all NER participants and organizes the correct NER repair complex. In the absence of XPA's scaffolding function, no repair process occurs. In this review, we briefly summarize our current knowledge about the XPA protein structure and analyze the formation of contact with its protein partners during NER complex assembling. We focus on different ways of regulation of the XPA protein's activity and expression and pay special attention to the network of post-translational modifications. We also discuss the data that is not in line with the currently accepted hypothesis about the functioning of the XPA protein.

Probing Protein-DNA Conformational Dynamics in DNA Damage Recognition: Xeroderma Pigmentosum Group A Stabilizes the Damaged DNA-RPA14 Complex by Controlling Conformational Fluctuation Dynamics.

DNA damage inside biological systems may result in diseases like cancer. One of the major repairing mechanisms is the nucleotide excision repair (NER) that recognizes and repairs the damage caused by several internal and external exposures, such as DNA double-strand distortion due to the chemical modifications. Recognition of lesions is the initial stage of the DNA damage repair, which occurs with the help of several proteins like Replication Protein A (RPA) and Xeroderma Pigmentosum group A (XPA). The recognition process involves complex conformational dynamics of the proteins. Studying the dynamics of damage recognition by these proteins helps us to understand the mechanism and to develop therapeutics to increase the efficiency of recognition. Here, we use single-molecule fluorescence fluctuation measurements of a dye, labeled at a damaged position on DNA, to understand the interaction of the damage site with RPA14 and XPA. Our results suggest that interactive conformational dynamics of RPA14 with damaged DNA is inhomogeneous due to its low affinity for DNA, whereas binding of XPA with the already formed DNA-RPA14 complex may increase the specificity of damage recognition by controlling the conformational fluctuation dynamics of the complex.

The Reactions of H2O2 and GSNO with the Zinc Finger Motif of XPA. Not A Regulatory Mechanism, But No Synergy with Cadmium Toxicity.

Tetrathiolate zinc fingers are potential targets of oxidative assault under cellular stress conditions. We used the synthetic 37-residue peptide representing the tetrathiolate zinc finger domain of the DNA repair protein XPA, acetyl-DYVICEECGKEFMSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf) as a working model to study the reaction of its Zn(II) complex (ZnXPAzf) with hydrogen peroxide and S-nitrosoglutathione (GSNO), as oxidative and nitrosative stress agents, respectively. We also used the Cd(II) substituted XPAzf (CdXPAzf) to assess the situation of cadmium assault, which is accompanied by oxidative stress. Using electrospray mass spectrometry (ESI-MS), HPLC, and UV-vis and circular dichroism spectroscopies we demonstrated that even very low levels of H2O2 and GSNO invariably cause irreversible thiol oxidation and concomitant Zn(II) release from ZnXPAzf. In contrast, CdXPAzf was more resistant to oxidation, demonstrating the absence of synergy between cadmium and oxidative stresses. Our results indicate that GSNO cannot act as a reversible modifier of XPA, and rather has a deleterious effect on DNA repair.

Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search.

Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by ∼60° as a monomer. Furthermore, we observe XPA specificity for the helix-distorting base adduct N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene over non-damaged dsDNA. Moreover, single molecule fluorescence microscopy reveals that DNA-bound XPA exhibits multiple modes of linear diffusion between paused phases. The presence of DNA damage increases the frequency of pausing. Truncated XPA, lacking the intrinsically disordered N- and C-termini, loses specificity for DNA lesions and shows less pausing on damaged DNA. Our data are consistent with a working model in which monomeric XPA bends DNA, displays episodic phases of linear diffusion along DNA, and pauses in response to DNA damage.

New structural insights into the recognition of undamaged splayed-arm DNA with a single pair of non-complementary nucleotides by human nucleotide excision repair protein XPA.

XPA (Xeroderma pigmentosum complementation group A) is a core scaffold protein that plays significant roles in DNA damage verification and recruiting downstream endonucleases in the nucleotide excision repair (NER) pathway. Here, we present the 2.81 Å resolution crystal structure of the DNA-binding domain (DBD) of human XPA in complex with an undamaged splayed-arm DNA substrate with a single pair of non-complementary nucleotides. The structure reveals that two XPA molecules bind to one splayed-arm DNA with a 10-bp duplex recognition motif in a non-sequence-specific manner. XPA molecules bind to both ends of the DNA duplex region with a characteristic ß-hairpin. A conserved tryptophan residue Trp175 packs against the last base pair of DNA duplex and stabilizes the conformation of the characteristic ß-hairpin. Upon DNA binding, the C-terminal last helix of XPA would shift towards the minor groove of the DNA substrate for better interaction. Notably, human XPA is able to bind to the undamaged DNA duplex without any kinks, and XPA-DNA binding does not bend the DNA substrate obviously. This study provides structural basis for the binding mechanism of XPA to the undamaged splayed-arm DNA with a single pair of non-complementary nucleotides.

A key interaction with RPA orients XPA in NER complexes.

The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD-RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.

High-resolution cryo-EM structures of TFIIH and their functional implications.

Eukaryotic transcription factor IIH (TFIIH) is a 500 kDa-multiprotein complex that harbors two SF2-family DNA-dependent ATPase/helicase subunits and the kinase activity of Cyclin-dependent kinase 7. TFIIH serves as a general transcription factor for transcription initiation by eukaryotic RNA polymerase II and plays an important role in nucleotide excision DNA repair. Aiming to understand the molecular mechanisms of its function and regulation in two key cellular pathways, the high-resolution structure of TFIIH has been pursued for decades. Recent breakthroughs, largely enabled by methodological advances in cryo-electron microscopy, have finally revealed the structure of TFIIH and its interactions in the context of the Pol II-pre-initiation complex, and provide a first glimpse of a TFIIH-containing assembly in DNA repair. Here, we review and discuss these recent structural insights and their functional implications.

Structural characterization of the redefined DNA-binding domain of human XPA.

XPA (xeroderma pigmentosum complementation group A), a key scaffold protein in nucleotide excision repair (NER) pathway, is important in DNA damage verification and repair proteins recruitment. Earlier studies had mapped the minimal DNA-binding domain (MBD) of XPA to a region corresponding to residues 98-219. However, recent studies indicated that the region involving residues 98-239 is the redefined DNA-binding domain (DBD), which binds to DNA substrates with a much higher binding affinity than MBD and possesses a nearly identical binding affinity to the full-length XPA protein. However, the structure of the redefined DBD domain of XPA (XPA-DBD) remains to be investigated. Here, we present the crystal structure of XPA-DBD at 2.06 Šresolution. Structure of the C-terminal region of XPA has been extended by 21 residues (Arg211-Arg231) as compared with previously reported MBD structures. The structure reveals that the C-terminal extension (Arg211-Arg231) is folded as an α-helix with multiple basic residues. The positively charged surface formed in the last C-terminal helix suggests its critical role in DNA binding. Further structural analysis demonstrates that the last C-terminal region (Asp217-Thr239) of XPA-DBD might undergo a conformational change to directly bind to the DNA substrates. This study provides a structural basis for understanding the possible mechanism of enhanced DNA-binding affinity of XPA-DBD.

Green Fluorescent Probe for Imaging His6-Tagged Proteins Inside Living Cells.

Small molecule-based fluorescent probes offer great opportunities for specifically tracking proteins in living systems with minimal perturbation on the protein function and localization. Herein, we report a small green fluorescent probe (Ni2+- NTA-AF) consisting of a Ni2+-NTA moiety, a fluorescein, and an arylazide group, that binds specifically to His6-tagged proteins with fluorescence enhancement in vitro upon photoactivation of the arylazide group. Importantly, the probe can cross the cell membranes and stoichiometrically label His6-tagged proteins rapidly (∼15 min) in living prokaryotic and eukaryotic cells exemplified by a DNA repair protein Xeroderma pigmentosum group A (XPA). Using the probe, we successfully visualized Sirtuin 5, which is localized to the mitochondria. This probe exhibits high quantum yields and improved solubility, offering a new opportunity for imaging intracellular His6-tagged proteins inside living cells with better contrast.

The redefined DNA-binding domain of human xeroderma pigmentosum complementation group A: production, crystallization and structure solution.

Human xeroderma pigmentosum complementation group A (XPA) is a scaffold protein that plays significant roles in DNA-damage verification and in recruiting downstream endonucleases to facilitate the repair of DNA lesions in nucleotide-excision repair. XPA98-219 (residues 98-219) has been identified as a DNA-binding domain and has been extensively studied in the last two decades. However, the most recent studies have redefined the DNA-binding domain as XPA98-239 (residues 98-239); it exerts a remarkably higher DNA-binding affinity than XPA98-219 and has a binding affinity that is quite similar to that of the full-length protein. Here, the production, crystallization and structure solution of human XPA98-239 are described. Crystals were obtained using a precipitant composed of 1.8 M ammonium citrate tribasic pH 7.0. Native X-ray diffraction data and zinc single-wavelength anomalous diffraction (SAD) data were collected to 1.93 and 2.06 Šresolution, respectively. The crystals belonged to space group P3, with unit-cell parameters a = 67.1, b = 67.1, c = 35.6 Å, γ = 120.0°. Crystal-content analysis showed the presence of one molecule in the asymmetric unit, corresponding to a Matthews coefficient of 2.65 Å3 Da-1 and a solvent content of 53.6%. The initial phases were solved and the structure model was automatically built by zinc SAD using the AutoSol program. The initial structure model covered 119 of 142 residues in the asymmetric unit, with an Rwork of 22.15% and an Rfree of 25.82%. Compared with a previously obtained truncated solution NMR structure of XPA (residues 98-210), a 19-residue C-terminal extension (residues 211-229, corresponding to 10 of the 20 extra C-terminal residues in the redefined domain for enhanced DNA binding) was contained in this initial model. Refinement of the atomic coordinates of XPA is ongoing.

Investigation of the probable homo-dimer model of the Xeroderma pigmentosum complementation group A (XPA) protein to represent the DNA-binding core.

The Xeroderma pigmentosum complementation group A (XPA) protein functions as a primary damage verifier and as a scaffold protein in nucleotide excision repair (NER) in all higher organisms. New evidence of XPA's existence as a dimer and the redefinition of its DNA-binding domain (DBD) raises new questions regarding the stability and functional position of XPA in NER. Here, we have investigated XPA's dimeric status with respect to its previously defined DBD (XPA98-219) as well as with its redefined DBD (XPA98-239). We studied the stability of XPA98-210 and XPA98-239 homo-dimer systems using all-atom molecular dynamics simulation, and we have also characterized the protein-protein interactions (PPI) of these two homo-dimeric forms of XPA. After conducting the root mean square deviation (RMSD) analyses, it was observed that the XPA98-239 homo-dimer has better stability than XPA98-210. It was also found that XPA98-239 has a larger number of hydrogen bonds, salt bridges, and hydrophobic interactions than the XPA98-210 homo-dimer. We further found that Lys, Glu, Gln, Asn, and Arg residues shared the major contribution toward the intermolecular interactions in XPA homo-dimers. The binding free energy (BFE) analysis, which used the molecular mechanics Poisson-Boltzmann method (MM-PBSA) and the generalized Born and surface area continuum solvation model (GBSA) for both XPA homo-dimers, also substantiated the positive result in favor of the stability of the XPA98-239 homo-dimer. Communicated by Ramaswamy H. Sarma.

Sirtuin 1-mediated deacetylation of XPA DNA repair protein enhances its interaction with ATR protein and promotes cAMP-induced DNA repair of UV damage.

Blunted melanocortin 1 receptor (MC1R) signaling promotes melanocyte genomic instability in part by attenuating cAMP-mediated DNA repair responses, particularly nucleotide excision repair (NER), which recognizes and clears mutagenic photodamage. cAMP-enhanced NER is mediated by interactions between the ataxia telangiectasia-mutated and Rad3-related (ATR) and xeroderma pigmentosum complementation group A (XPA) proteins. We now report a critical role for sirtuin 1 (SIRT1) in regulating ATR-mediated phosphorylation of XPA. SIRT1 deacetylates XPA at residues Lys-63, Lys-67, and Lys-215 to promote interactions with ATR. Mutant XPA containing acetylation mimetics at residues Lys-63, Lys-67, and Lys-215 exhibit blunted UV-dependent ATR-XPA interactions even in the presence of cAMP signals. ATR-mediated phosphorylation of XPA on Ser-196 enhances cAMP-mediated optimization of NER and is promoted by SIRT1-mediated deacetylation of XPA on Lys-63, Lys-67, and Lys-215. Interference with ATR-mediated XPA phosphorylation at Ser-196 by persistent acetylation of XPA at Lys-63, Lys-67, and Lys-215 delays repair of UV-induced DNA damage and attenuates cAMP-enhanced NER. Our study identifies a regulatory ATR-SIRT1-XPA axis in cAMP-mediated regulation melanocyte genomic stability, involving SIRT1-mediated deacetylation (Lys-63, Lys-67, and Lys-215) and ATR-dependent phosphorylation (Ser-196) post-translational modifications of the core NER factor XPA.

Structural dynamics and interactions of Xeroderma pigmentosum complementation group A (XPA98-210) with damaged DNA.

Nucleotide excision repair (NER) in higher organisms repair massive DNA abrasions caused by ultraviolet rays, and various mutagens, where Xeroderma pigmentosum group A (XPA) protein is known to be involved in damage recognition step. Any mutations in XPA cause classical Xeroderma pigmentosum disease. The extent to which XPA is required in the NER is still unclear. Here, we present the comparative study on the structural and conformational changes in globular DNA binding domain of XPA98-210 in DNA bound and DNA free state. Atomistic molecular dynamics simulation was carried out for both XPA98-210 systems using AMBER force fields. We observed that XPA98-210 in presence of damaged DNA exhibited more structural changes compared to XPA98-210 in its free form. When XPA is in contact with DNA, we found marked stability of the complex due to the formation of characteristic longer antiparallel ß-sheets consisting mainly lysine residues.

Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.

Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients.

Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN.

Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled by several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.

Kinetics and thermodynamics of zinc(II) and arsenic(III) binding to XPA and PARP-1 zinc finger peptides.

Inhibition of DNA repair is an established mechanism of arsenic co-carcinogenesis, and may be perpetuated by the binding of As(III) to key zinc finger (zf) DNA repair proteins. Validated molecular targets of As(III) include the first zinc finger domain of Poly (ADP-Ribose) Polymerase 1 (PARP-1), and the zinc finger domain of Xeroderma Pigmentosum Complementation Group A (XPA). In order to gain an understanding of the thermodynamic and kinetic parameters of the interaction of As(III) with these two zinc finger motifs, a fluorescence based approach was used to investigate Zn(II) and As(III) binding to synthetic model peptides corresponding to the zf motif of XPA and first zf motif of PARP-1, referred to in this paper as XPAzf and PARP-1zf-1, respectively. While XPAzf and PARP-1zf-1 display similar relative affinities for As(III), PARP-1zf-1 shows a potential kinetic advantage over XPAzf for As(III) binding, with a rate constant for the fast phase of formation of As(III)-PARP-1zf-1 approximately 4-fold higher than for As(III)-XPAzf. However, the binding of Zn(II) with either peptide proceeds at a faster rate than As(III). Notably, XPAzf demonstrates comparable affinities for binding both metals, while PARP-1zf-1 shows a slightly higher affinity for Zn(II), suggesting that the relative concentrations of Zn(II) and As(III) in a system may significantly influence which species predominates in zinc finger occupancy. These results provide insight into the mechanisms underlying interactions between zinc finger structures and As(III), and highlight the potential utility of zinc supplementation in mitigating adverse effects of As(III) on zinc finger functions in vivo.

XPA: A key scaffold for human nucleotide excision repair.

Nucleotide excision repair (NER) is essential for removing many types of DNA lesions from the genome, yet the mechanisms of NER in humans remain poorly understood. This review summarizes our current understanding of the structure, biochemistry, interaction partners, mechanisms, and disease-associated mutations of one of the critical NER proteins, XPA.

Metal binding mediated conformational change of XPA protein:a potential cytotoxic mechanism of nickel in the nucleotide excision repair.

Nucleotide excision repair (NER) is a pivotal life process for repairing DNA nucleotide mismatch caused by chemicals, metal ions, radiation, and other factors. As the initiation step of NER, the xeroderma pigmentosum complementation group A protein (XPA) recognizes damaged DNA molecules, and recruits the replication protein A (RPA), another important player in the NER process. The stability of the Zn(2+)-chelated Zn-finger domain of XPA center core portion (i.e., XPA98-210) is the foundation of its biological functionality, while the displacement of the Zn(2+) by toxic metal ions (such as Ni(2+), a known human carcinogen and allergen) may impair the effectiveness of NER and hence elevate the chance of carcinogenesis. In this study, we first calculated the force field parameters for the bonded model in the metal center of the XPA98-210 system, showing that the calculated results, including charges, bonds, angles etc., are congruent with previously reported results measured by spectrometry experiments and quantum chemistry computation. Then, comparative molecular dynamics simulations using these parameters revealed the changes in the conformation and motion mode of XPA98-210 Zn-finger after the substitution of Zn(2+) by Ni(2+). The results showed that Ni(2+) dramatically disrupted the relative positions of the four Cys residues in the Zn-finger structure, forcing them to collapse from a tetrahedron into an almost planar structure. Finally, we acquired the binding mode of XPA98-210 with its ligands RPA70N and DNA based on molecular docking and structural alignment. We found that XPA98-210's Zn-finger domain primarily binds to a V-shaped cleft in RPA70N, while the cationic band in its C-terminal subdomain participates in the recognition of damaged DNA. In addition, this article sheds light on the multi-component interaction pattern among XPA, DNA, and other NER-related proteins (i.e., RPA70N, RPA70A, RPA70B, RPA70C, RPA32, and RPA14) based on previously reported structural biology information. Thus, we derived a putative cytotoxic mechanism associated with the nickel ion, where the Ni(2+) disrupts the conformation of the XPA Zn-finger, directly weakening its interaction with RPA70N, and thus lowering the effectiveness of the NER process. In sum, this work not only provides a theoretical insight into the multi-protein interactions involved in the NER process and potential cytotoxic mechanism associated with Ni(2+) binding in XPA, but may also facilitate rational anti-cancer drug design based on the NER mechanism.