Eukaryotic Nucleotide Excision Repair Research Articles

Abstract 2106: Combined deacetylase and bromodomain inhibition downregulates ERCC2 and suppresses growth of metastatic colon cancer cells

Abstract DNA repair genes have potential clinical value in predicting cancer prognosis and treatment outcomes. Nucleotide excision repair (NER) proteins like ERCC2 play a critical role in maintaining genome integrity by recognizing and unwinding DNA at sites of damage1.Aberrant expression of ERCC2 alters NER capacity, influencing treatment outcomes2. The current investigation examined the expression of ERCC2 following epigenetic combination treatment in colorectal cancer (CRC) cells and preclinical models. Attention was drawn to ERCC2 based on three observations. First, from online databases, when ERCC2 was overexpressed in colon tumors the corresponding CRC patients had reduced overall survival3. Second, ERCC2 was the most highly downregulated gene when dietary histone deacetylase 3 (HDAC3) inhibitor sulforaphane (SFN) was combined withJQ1, an inhibitor of the bromodomain and extra terminal domain family, in human colon cancer cells and in colon polyps from the polyposis in rat colon (Pirc) model4. Third, as reported here for the first time, RNA-seq analyses of Pirc colon polyps from rats treated withJQ1 and the SFN analog 6-SFN5 identified Ercc2 as the most highly downregulated gene. RNA-seq data were corroborated by RT-qPCR and immunoblotting experiments. There is much interest in combination approaches that target epigenetic ‘readers, writers, and erasers' that are deregulated in cancer and other pathologies. The current work identified promising second-generation inhibitors with enhanced synergy and antitumor efficacy, especially in metastatic cells cultured in three-dimensions. Drug combinations decreased HDAC3, BRD4 and ERCC2 expression, while DNA damage and apoptosis markers were increased both in spheroids and in a mouse xenograft model. This investigation has potential clinical relevance for the identification of robust biomarkers that predict enhanced antitumor outcomes in CRC patients.1. Gillet LC, Schärer OD. Molecular mechanisms of mammalian global genome nucleotide excision repair. Chem Rev 2006;106:253-76.2. Shuck SC, Short EA, Turchi JJ. Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology. Cell Res 2008;18:64-72.3. Liu J et al., The Differential Expression of Core Genes in Nucleotide Excision Repair Pathway Indicates Colorectal Carcinogenesis and Prognosis. Biomed Res Int 2018;2018:9651320.4. Rajendran P et al., Acetylation of CCAR2 establishes a BET/BRD9 acetyl switch in response to combined deacetylase and bromodomain inhibition. Cancer Res 2019;79:918-27.5. Rajendran Pet al., HDAC turnover, CtIP acetylation and dysregulated DNA damage signaling in colon cancer cell treated with sulforaphane and related dietary isothiocyanates. Epigenetics 2013;8:612-23. Citation Format: Sabeeta Kapoor, Trace Gustafson, Mutian Zhang, Ying-Shiuan Chen, Jia Li, Nhung Nguyen, Jorge Enrique Perez, Wan Mohaiza Dashwood, Praveen Rajendran, Roderick Dashwood. Combined deacetylase and bromodomain inhibition downregulates ERCC2 and suppresses growth of metastatic colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2106.

Tethering-facilitated DNA 'opening' and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein.

XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively ‘opening’ these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a ‘kinetic gating’ mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how ‘opening’ is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed ‘open’ undamaged DNA in solution and that such ‘opening’ can largely occur without one or the other of the β-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound ‘open’ DNA adopts multiple conformations in solution notwithstanding the DNA’s original structure or the β-hairpins. Molecular dynamics simulations reveal compensatory roles of the β-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA ‘opening’ of undamaged sites and the dynamic nature of ‘open’ DNA may shed light on how the protein functions within and beyond nucleotide excision repair in cells.

Open gaps in the evolution of the eukaryotic nucleotide excision repair

Nucleotide excision repair (NER) is the most versatile DNA repair pathway as it removes different kinds of bulky lesions. Due to its essential role for genome integrity, it has appeared early in the evolution of species. However, most published studies are focused on humans, mice, yeast or bacteria. Considering the large amount of information on genome databases, it is currently possible to retrieve sequences from NER components in many organisms. Therefore, we have characterized the potential orthologs of 10 critical components of the human NER pathway in 12 eukaryotic species by using similarity and structural criteria through the use of bioinformatics tools. This approach has allowed us to characterize gene and protein structures comparatively, taking a glance at some evolutionary aspects of the NER pathway. We have obtained significant search results for the majority of the proteins in most of the organisms studied, mainly for factors that play a pivotal role in the pathway. However, we have revisited significant differences and found new aspects that may imply a distinct functioning of this pathway in different organisms. Through the demonstration of the heterogeneity of the gene structures and a variety in the protein architecture of the NER components evaluated, our results show important differences between human NER and evolutionarily distant eukaryotes. We highlight the lack of a canonical XPD in chicken, the divergence of XPA in plants and protozoans and the absence of XPE in the invertebrate species analyzed. In spite of this, it is remarkable the presence of this excision repair mechanism in a high number of evolutionary distant organisms, being present since the origin of eukaryotes.

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.

Mercury (II) impairs nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos by targeting primarily at the stage of DNA incision

Mercuric ion (Hg2+) is the most prevalent form of inorganic Hg found in polluted aquatic environment. As inhibition of DNA damage repair has been proposed as one of the mechanisms of Hg2+-induced genotoxicity in aquatic animals and mammalian cells, this study explored the susceptibility of different stages of nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos to Hg2+ using UV-damaged DNA as the repair substrate. Exposure of embryos at 1h post fertilization (hpf) to HgCl2 at 0.1–2.5μM for 9h caused a concentration-dependent inhibition of NER capacity monitored by a transcription-based DNA repair assay. The extracts of embryos exposed to 2.5μM Hg2+ almost failed to up-regulate UV-suppressed marker cDNA transcription. No inhibition of ATP production was observed in all Hg2+-exposed embryos. Hg2+ exposure imposed either weak inhibitory or stimulating effects on the gene expression of NER factors, while band shift assay showed the inhibition of photolesion binding activities to about 40% of control in embryos treated with 1–2.5μM HgCl2. The damage incision stage of NER in zebrafish embryos was found to be more sensitive to Hg2+ than photolesion binding capacity due to the complete loss of damage incision activity in the extracts of embryos exposed to 1–2.5μM Hg2+. NER-related DNA incision was induced in UV-irradiated embryos based on the production of short DNA fragments matching the sizes of excision products generated by eukaryotic NER. Pre-exposure of embryos to Hg2+ at 0.1–2.5μM all suppressed DNA incision/excision in UV-irradiated embryos, reflecting a high sensitivity of DNA damage incision/excision to Hg2+. Our results showed the potential of Hg2+ at environmental relevant levels to disturb NER in zebrafish embryos by targeting primarily at the stage of DNA incision/excision.

The rad2 gene of haloarchaeum Halobacterium salinarum is functional in the repair of ultraviolet light induced DNA photoproducts

There are a lot of bacterial and eukaryotic DNA repair gene homologs among sequenced archaeal genomes but there is little information about DNA repair mechanisms and the interaction of involved repair proteins. In order to study DNA repair mechanisms in the third domain of life, we studied these processes in the model archaeon, Halobacterium salinarum. H. salinarum has homologs of eukaryotic nucleotide excision repair genes such as rad2 gene. A functional analysis of rad2 was performed by knocking down of this gene. We introduced an antisense RNA expression vector into the cells and the sensitivity of transformants against ultraviolet light exposure was measured to determine whether rad2 gene performs any role in the repair of the DNA lesions induced by UV light or not. Our data suggests that rad2 is functional in this pathway and knocked down strains were unable to completely repair the UV induced DNA damages. In this study, for the first time antisense RNA is used for functional analysis of a gene in H. salinarum and it is shown that antisense RNA could be used as a reliable genetic tool for understanding of the archaeal genetics.

Structure and specificity of FEN-1 from Methanopyrus kandleri.

DNA repair is fundamental to genome stability and is found in all three domains of life. However many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologs, and those present often contain significant differences compared to their eukaryotic homologs. To clarify the role of the NER XPG-like protein Mk0566 from M. kandleri, its biochemical activity and three-dimensional structure were investigated. Both were found to be more similar to human FEN-1 than human XPG, suggesting a biological role in replication and long-patch base excision repair rather than in NER.

Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5'-3' helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a "final" verification state, which may then trigger the recruitment of further NER proteins.

Ribonucleotides as nucleotide excision repair substrates

The incorporation of ribonucleotides in DNA has attracted considerable notice in recent years, since the pool of ribonucleotides can exceed that of the deoxyribonucleotides by at least 10-20-fold, and single ribonucleotide incorporation by DNA polymerases appears to be a common event. Moreover ribonucleotides are potentially mutagenic and lead to genome instability. As a consequence, errantly incorporated ribonucleotides are rapidly repaired in a process dependent upon RNase H enzymes. On the other hand, global genomic nucleotide excision repair (NER) in prokaryotes and eukaryotes removes damage caused by covalent modifications that typically distort and destabilize DNA through the production of lesions derived from bulky chemical carcinogens, such as polycyclic aromatic hydrocarbon metabolites, or via crosslinking. However, a recent study challenges this lesion-recognition paradigm. The work of Vaisman et al. (2013) [34] reveals that even a single ribonucleotide embedded in a deoxyribonucleotide duplex is recognized by the bacterial NER machinery in vitro. In their report, the authors show that spontaneous mutagenesis promoted by a steric-gate pol V mutant increases in uvrA, uvrB, or uvrC strains lacking rnhB (encoding RNase HII) and to a greater extent in an NER-deficient strain lacking both RNase HI and RNase HII. Using purified UvrA, UvrB, and UvrC proteins in in vitro assays they show that despite causing little distortion, a single ribonucleotide embedded in a DNA duplex is recognized and doubly-incised by the NER complex. We present the hypothesis to explain the recognition and/or verification of this small lesion, that the critical 2'-OH of the ribonucleotide - with its unique electrostatic and hydrogen bonding properties - may act as a signal through interactions with amino acid residues of the prokaryotic NER complex that are not possible with DNA. Such a mechanism might also be relevant if it were demonstrated that the eukaryotic NER machinery likewise incises an embedded ribonucleotide in DNA.

XPF-ERCC1: On the Bubble

In this issue of Structure, Das et al. report the structure of the helix-hairpin-helix dimerization domain of XPF bound to ssDNA. These results provide insight into the architecture of nucleotide excision repair machinery and how it interacts with damaged DNA substrates.

Surviving the Sun: Repair and bypass of DNA UV lesions

Structural studies of UV-induced lesions and their complexes with repair proteins reveal an intrinsic flexibility of DNA at lesion sites. Reduced DNA rigidity stems primarily from the loss of base stacking, which may manifest as bending, unwinding, base unstacking, or flipping out. The intrinsic flexibility at UV lesions allows efficient initial lesion recognition within a pool of millions to billions of normal DNA base pairs. To bypass the damaged site by translesion synthesis, the specialized DNA polymerase η acts like a molecular "splint" and reinforces B-form DNA by numerous protein-phosphate interactions. Photolyases and glycosylases that specifically repair UV lesions interact directly with UV lesions in bent DNA via surface complementation. UvrA and UvrB, which recognize a variety of lesions in the bacterial nucleotide excision repair pathway, appear to exploit hysteresis exhibited by DNA lesions and conduct an ATP-dependent stress test to distort and separate DNA strands. Similar stress tests are likely conducted in eukaryotic nucleotide excision repair.

Probing for DNA damage with β-hairpins: Similarities in incision efficiencies of bulky DNA adducts by prokaryotic and human nucleotide excision repair systems in vitro

Nucleotide excision repair (NER) is an important prokaryotic and eukaryotic defense mechanism that removes a large variety of structurally distinct lesions in cellular DNA. While the proteins involved are completely different, the mode of action of these two repair systems is similar, involving a cut-and-patch mechanism in which an oligonucleotide sequence containing the lesion is excised. The prokaryotic and eukaryotic NER damage-recognition factors have common structural features of β-hairpin intrusion between the two DNA strands at the site of the lesion. In the present study, we explored the hypothesis that this common β-hairpin intrusion motif is mirrored in parallel NER incision efficiencies in the two systems. We have utilized human HeLa cell extracts and the prokaryotic UvrABC proteins to determine their relative NER incision efficiencies. We report here comparisons of relative NER efficiencies with a set of stereoisomeric DNA lesions derived from metabolites of benzo[ a]pyrene and equine estrogens in different sequence contexts, utilizing 21 samples. We found a general qualitative trend toward similar relative NER incision efficiencies for ∼65% of these substrates; the other cases deviate mostly by ∼30% or less from a perfect correlation, although several more distant outliers are also evident. This resemblance is consistent with the hypothesis that lesion recognition through β-hairpin insertion, a common feature of the two systems, is facilitated by local thermodynamic destabilization induced by the lesions in both cases. In the case of the UvrABC system, varying the nature of the UvrC endonuclease, while maintaining the same UvrA/B proteins, can markedly affect the relative incision efficiencies. These observations suggest that, in addition to recognition involving the initial modified duplexes, downstream events involving UvrC can also play a role in distinguishing and processing different lesions in prokaryotic NER.

Regulation and Rate Enhancement during Transcription-Coupled DNA Repair

SummaryTranscription-coupled DNA repair (TCR) is a subpathway of nucleotide excision repair (NER) that is triggered when RNA polymerase is stalled by DNA damage. Lesions targeted by TCR are repaired more quickly than lesions repaired by the transcription-independent “global” NER pathway, but the mechanism underlying this rate enhancement is not understood. Damage recognition during bacterial NER depends upon UvrA, which binds to the damage and loads UvrB onto the DNA. Bacterial TCR additionally requires the Mfd protein, a DNA translocase that removes the stalled transcription complexes. We have determined the properties of Mfd, UvrA, and UvrB that are required for the elevated rate of repair observed during TCR. We show that TCR and global NER differ in their requirements for damage recognition by UvrA, indicating that Mfd acts at the very earliest stage of the repair process and extending the functional similarities between TCR in bacteria and eukaryotes.

SIRT1 Regulates UV-Induced DNA Repair through Deacetylating XPA

SIRT1, a NAD(+)-dependent histone deacetylase, plays crucial roles in multiple biological processes including gene transcription, cellular metabolism, stress response, and tumorigenesis. Xeroderma pigmentosum group A (XPA) is a core nucleotide excision repair (NER) factor essential for NER process. Here we show that SIRT1 plays an important role in the regulation of NER pathway. Downregulation of SIRT1 significantly sensitizes cells to UV irradiation. SIRT1 interacts with XPA, and the interaction is enhanced after UV irradiation. XPA can be acetylated at lysines 63 and 67. SIRT1 deacetylates XPA both in vitro and in cells. Importantly, SIRT1-mediated deacetylation of XPA is required for optimal NER pathway since XPA-deficient cells complemented with XPA-K6367Q, which mimics hyperacetylated XPA, display significantly higher UV sensitivity compared with the XPA cells complemented with wild-type XPA. Furthermore, SIRT1-mediated XPA deacetylation enhances its interaction with RPA32. Our results demonstrate that SIRT1 regulates NER pathway through modulation of XPA acetylation status.

Structure determination of the minimal complex between Tfb5 and Tfb2, two subunits of the yeast transcription/DNA-repair factor TFIIH: a retrospective study

Tfb5 interacts with the Tfb2 subunit of the general transcription factor TFIIH to ensure efficient nucleotide-excision repair in eukaryotes. The crystal structure of the complex between Tfb5 and the C-terminal region of Tfb2 (Tfb2C) from Saccharomyces cerevisiae has recently been reported. Here, the structure-determination process is described as a case study. Although crystals were obtained readily, it was not possible to determine experimental phases from a first crystal form (Tfb2(412-513)-Tfb5(2-72)) that diffracted to 2.6 A resolution. Shortening of the Tfb2C from its N-terminus was decisive and modified the crystal packing, leading to a second crystal form (Tfb2(435-513)-Tfb5(2-72)). These crystals diffracted to 1.7 A resolution with excellent mosaicity and allowed structure determination by conventional approaches using heavy atoms. The refined structure from the second crystal form was used to solve the structure of the first crystal form by molecular replacement. Comparison of the two structures revealed that the N-terminal region of Tfb2C and (to a lesser extent) the C-terminal region of Tfb5 contributed to the crystal packing. A detailed analysis illustrates how variation in domain boundaries influences crystal packing and quality.

Involvement of Global Genome Repair, Transcription Coupled Repair, and Chromatin Remodeling in UV DNA Damage Response Changes during Development

Nucleotide Excision Repair (NER), which removes a variety of helix-distorting lesions from DNA, is initiated by two distinct DNA damage-sensing mechanisms. Transcription Coupled Repair (TCR) removes damage from the active strand of transcribed genes and depends on the SWI/SNF family protein CSB. Global Genome Repair (GGR) removes damage present elsewhere in the genome and depends on damage recognition by the XPC/RAD23/Centrin2 complex. Currently, it is not well understood to what extent both pathways contribute to genome maintenance and cell survival in a developing organism exposed to UV light. Here, we show that eukaryotic NER, initiated by two distinct subpathways, is well conserved in the nematode Caenorhabditis elegans. In C. elegans, involvement of TCR and GGR in the UV-induced DNA damage response changes during development. In germ cells and early embryos, we find that GGR is the major pathway contributing to normal development and survival after UV irradiation, whereas in later developmental stages TCR is predominantly engaged. Furthermore, we identify four ISWI/Cohesin and four SWI/SNF family chromatin remodeling factors that are implicated in the UV damage response in a developmental stage dependent manner. These in vivo studies strongly suggest that involvement of different repair pathways and chromatin remodeling proteins in UV-induced DNA repair depends on developmental stage of cells.

Bax1 Is a Novel Endonuclease

The helicases XPB and XPD are part of the TFIIH complex, which mediates transcription initiation as well as eukaryotic nucleotide excision repair (NER). Although there is no TFIIH complex present in archaea, most species contain both XPB and XPD and serve as a model for their eukaryotic homologs. Recently, a novel binding partner for XPB, Bax1 (binds archeal XPB), was identified in archaea. To gain insights into its role in NER, Bax1 from Thermoplasma acidophilum was characterized. We identified Bax1 as a novel Mg(2+)-dependent structure-specific endonuclease recognizing DNA containing a 3' overhang. Incision assays conducted with DNA substrates providing different lengths of the 3' overhang indicate that Bax1 specifically incises DNA in the single-stranded region of the 3' overhang 4-6 nucleotides to the single-stranded DNA/double-stranded DNA junction and thus is a structure-specific and not a sequence-specific endonuclease. In contrast, no incision was detected in the presence of a 5' overhang, double-stranded DNA, or DNA containing few unpaired nucleotides forming a bubble. Several Bax1 variants were generated based on multiple sequence alignments and examined with respect to their ability to perform the incision reaction. Residues Glu-124, Asp-132, Tyr-152, and Glu-155 show a dramatic reduction in incision activity, indicating a pivotal role in catalysis. Interestingly, Bax1 does not exhibit any incision activity in the presence of XPB, thus suggesting a role in NER in which the endonuclease activity is tightly regulated until the damage has been recognized and verified prior to the incision event.

Comparison of the activities of bacterial and mammalian nucleotide excision repair systems for DNA-protein crosslinks

Endogenous and environmental genotoxic agents produce DNA damage and induce cell death and mutations. The repair mechanisms of base lesions and single and double strand breaks have been well characterized in both prokaryotic and eukaryotic cells. However, the molecular pathways that repair or tolerate DNA-protein crosslinks (DPCs) remains to be largely elucidated. In this study, we constructed DNA substrates containing defined DPCs and assessed the incision activities of prokaryotic and eukaryotic nucleotide excision repair systems for DPCs in vitro.

Structure of the DNA Repair Helicase XPD

The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalyzing DNA duplex opening localized to the transcription start site or site of DNA damage, respectively. XPD has a 5' to 3' polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigmentosum, trichothiodystrophy, and Cockayne's syndrome, characterized by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 A crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans.

Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology

Repair of bulky DNA adducts by the nucleotide excision repair (NER) pathway is one of the more versatile DNA repair pathways for the removal of DNA lesions. There are two subsets of the NER pathway, global genomic-NER (GG-NER) and transcription-coupled NER (TC-NER), which differ only in the step involving recognition of the DNA lesion. Following recognition of the damage, the sub-pathways then converge for the incision/excision steps and subsequent gap filling and ligation steps. This review will focus on the GGR sub-pathway of NER, while the TCR sub-pathway will be covered in another article in this issue. The ability of the NER pathway to repair a wide array of adducts stems, in part, from the mechanisms involved in the initial recognition step of the damaged DNA and results in NER impacting an equally wide array of human physiological responses and events. In this review, the impact of NER on carcinogenesis, neurological function, sensitivity to environmental factors and sensitivity to cancer therapeutics will be discussed. The knowledge generated in our understanding of the NER pathway over the past 40 years has resulted from advances in the fields of animal model systems, mammalian genetics and in vitro biochemistry, as well as from reconstitution studies and structural analyses of the proteins and enzymes that participate in this pathway. Each of these avenues of research has contributed significantly to our understanding of how the NER pathway works and how alterations in NER activity, both positive and negative, influence human biology.