Nucleotide Excision Repair Complex Research Articles

A compound heterozygous mutation of ERCC8 is responsible for a family with Cockayne syndrome.

Cockayne syndrome is an inherited heterogeneous defect in transcription-coupled DNA repair (TCR) cause severe clinical syndromes, which may affect the nervous system development of infants and even lead to premature death in some cases. ERCC8 diverse critical roles in the nucleotide excision repair (NER) complex, which is one of the disease-causing genes of Cockayne syndrome. The mutation of ERCC8 in the patient was identified and validated using WES and Sanger sequencing. Specifically, a compound heterozygous mutation (c.454_460dupGTCTCCA p. T154Sfs*13 and c.755_759delGTTTT p.C252Yfs*3) of ERCC8 (CSA) was found, which could potentially be the genetic cause of Cockayne syndrome in the proband. In this study, we identified a novel heterozygous mutation of ERCC8 in a Chinese family with Cockayne syndrome, which enlarging the genetic spectrum of the disease.

ERCC1 abundance is an indicator of DNA repair-apoptosis decision upon DNA damage

DNA repair is essential for successful propagation of genetic material and fidelity of transcription. Nucleotide excision repair (NER) is one of the earliest DNA repair mechanisms, functionally conserved from bacteria to human. The fact that number of NER genes vary significantly between prokaryotes and metazoans gives the insight that NER proteins have evolved to acquire additional functions to combat challenges associated with a diploid genome, including being involved in the decision between DNA repair and apoptosis. However, no direct association between apoptosis and NER proteins has been shown to date. In this study, we induced apoptosis with a variety of agents, including oxaliplatin, doxorubicin and TRAIL, and observed changes in the abundance and molecular weight of NER complex proteins. Our results showed that XPA, XPC and ERCC1 protein levels change during DNA damage-induced apoptosis. Among these, ERCC1 decrease was observed as a pre-mitochondria depolarisation event which marks the “point of no return” in apoptosis signalling. ERCC1 decrease was due to proteasomal degradation upon lethal doses of oxaliplatin exposure. When ERCC1 protein was stabilised using proteasome inhibitors, the pro-apoptotic activity of oxaliplatin was attenuated. These results explain why clinical trials using proteasome inhibitors and platinum derivatives showed limited efficacy in carcinoma treatment and also the importance of how deep understanding of DNA repair mechanisms can improve cancer therapy.

Facial clues to the photosensitive trichothiodystrophy phenotype in childhood.

Among genodermatoses, trichothiodystrophies (TTDs) are a rare genetically heterogeneous group of syndromic conditions, presenting with skin, hair, and nail abnormalities. An extra-cutaneous involvement (craniofacial district and neurodevelopment) can be also a part of the clinical picture. The presence of photosensitivity describes three forms of TTDs: MIM#601675 (TTD1), MIM#616390 (TTD2) and MIM#616395 (TTD3), that are caused by variants afflicting some components of the DNA Nucleotide Excision Repair (NER) complex and with more marked clinical consequences. In the present research, 24 frontal images of paediatric patients with photosensitive TTDs suitable for facial analysis through the next-generation phenotyping (NGP) technology were obtained from the medical literature. The pictures were compared to age and sex-matched to unaffected controls using 2 distinct deep-learning algorithms: DeepGestalt and GestaltMatcher (Face2Gene, FDNA Inc., USA). To give further support to the observed results, a careful clinical revision was undertaken for each facial feature in paediatric patients with TTD1 or TTD2 or TTD3. Interestingly, a distinctive facial phenotype emerged by the NGP analysis delineating a specific craniofacial dysmorphic spectrum. In addition, we tabulated every single detail within the observed cohort. The novelty of the present research includes the facial characterization in children with the photosensitive types of TTDs through the 2 different algorithms. This result can become additional criteria for early diagnosis, and for subsequent targeted molecular investigations as well as a possible tailored multidisciplinary personalized management.

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.

A Novel Missense Mutation in ERCC8 Co-Segregates with Cerebellar Ataxia in a Consanguineous Pakistani Family.

Autosomal-recessive cerebellar ataxias (ARCAs) are heterogeneous rare disorders mainly affecting the cerebellum and manifest as movement disorders in children and young adults. To date, ARCA causing mutations have been identified in nearly 100 genes; however, they account for less than 50% of all cases. We studied a multiplex, consanguineous Pakistani family presenting with a slowly progressive gait ataxia, body imbalance, and dysarthria. Cerebellar atrophy was identified by magnetic resonance imaging of brain. Using whole exome sequencing, a novel homozygous missense mutation ERCC8:c.176T>C (p.M59T) was identified that co-segregated with the disease. Previous studies have identified homozygous mutations in ERCC8 as causal for Cockayne Syndrome type A (CSA), a UV light-sensitive syndrome, and several ARCAs. ERCC8 plays critical roles in the nucleotide excision repair complex. The p.M59T, a substitution mutation, is located in a highly conserved WD1 beta-transducin repeat motif. In silico modeling showed that the structure of this protein is significantly affected by the p.M59T mutation, likely impairing complex formation and protein-protein interactions. In cultured cells, the p.M59T mutation significantly lowered protein stability compared to wildtype ERCC8 protein. These findings expand the role of ERCC8 mutations in ARCAs and indicate that ERCC8-related mutations should be considered in the differential diagnosis of ARCAs.

Two interaction surfaces between XPA and RPA organize the preincision complex in nucleotide excision repair

The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.

Saccharomyces cerevisiae DNA repair pathways involved in repair of lesions induced by mixed ternary mononuclear Cu(II) complexes based on valproic acid with 1,10-phenanthroline or 2,2’- bipyridine ligands

The sodium valproate has been largely used as an anti-epilepsy drug and, recently, as a putative drug in cancer therapy. However, the treatment with sodium valproate has some adverse effects. In this sense, more effective and secure complexes than sodium valproate should be explored in searching for new active drugs. This study aims to evaluate the cytotoxicity of sodium valproate, mixed ternary mononuclear Cu(II) complexes based on valproic acid (VA) with 1,10-phenanthroline (Phen) or 2,2’- bipyridine (Bipy) ligands - [Cu2(Valp)4], [Cu(Valp)2Phen] and [Cu(Valp)2Bipy] - in yeast Saccharomyces cerevisiae, proficient or deficient in different repair pathways, such as base excision repair (BER), nucleotide excision repair (NER), translesion synthesis (TLS), DNA postreplication repair (PRR), homologous recombination (HR) and non-homologous end-joining (NHEJ). The results indicated that the Cu(II) complexes have higher cytotoxicity than sodium valproate in the following order: [Cu(Valp)2Phen] > [Cu(Valp)2Bipy] > [Cu2(Valp)4] > sodium valproate. The treatment with Cu(II) complexes and sodium valproate induced mutations in S. cerevisiae. The data indicated that yeast strains deficient in BER (Ogg1p), NER (complex Rad1p-Rad10p) or TLS (Rev1p, Rev3p and Rad30p) proteins are associated with increased sensitivity to sodium valproate. The BER mutants (ogg1Δ, apn1Δ, rad27Δ, ntg1Δ and ntg2Δ) showed increased sensitivity to Cu(II) complexes. DNA damage induced by the complexes requires proteins from NER (Rad1p and Rad10p), TLS (Rev1p, Rev3p and Rad30p), PRR (Rad6 and Rad18p) and HR (Rad52p and Rad50p) for efficient repair. Therefore, Cu(II) complexes display enhanced cytotoxicity when compared to the sodium valproate and induce distinct DNA lesions, indicating a potential application as cytotoxic agents.

Impact of DNA sequences on DNA ‘opening’ by the Rad4/XPC nucleotide excision repair complex

Rad4/XPC recognizes diverse DNA lesions to initiate nucleotide excision repair (NER). However, NER propensities among lesions vary widely and repair-resistant lesions are persistent and thus highly mutagenic. Rad4 recognizes repair-proficient lesions by unwinding (‘opening’) the damaged DNA site. Such ‘opening’ is also observed on a normal DNA sequence containing consecutive C/G’s (CCC/GGG) when tethered to Rad4 to prevent protein diffusion. However, it was unknown if such tethering-facilitated DNA ‘opening’ could occur on any DNA or if certain structures/sequences would resist being ‘opened’. Here, we report that DNA containing alternating C/G’s (CGC/GCG) failed to be opened even when tethered; instead, Rad4 bound in a 180°-reversed manner, capping the DNA end. Fluorescence lifetime studies of DNA conformations in solution showed that CCC/GGG exhibits local pre-melting that is absent in CGC/GCG. In MD simulations, CGC/GCG failed to engage Rad4 to promote ‘opening’ contrary to CCC/GGG. Altogether, our study illustrates how local sequences can impact DNA recognition by Rad4/XPC and how certain DNA sites resist being ‘opened’ even with Rad4 held at that site indefinitely. The contrast between CCC/GGG and CGC/GCG sequences in Rad4-DNA recognition may help decipher a lesion’s mutagenicity in various genomic sequence contexts to explain lesion-determined mutational hot and cold spots.

Distinct DNA repair pathways cause genomic instability at alternative DNA structures

Alternative DNA structure-forming sequences can stimulate mutagenesis and are enriched at mutation hotspots in human cancer genomes, implicating them in disease etiology. However, the mechanisms involved are not well characterized. Here, we discover that Z-DNA is mutagenic in yeast as well as human cells, and that the nucleotide excision repair complex, Rad10-Rad1(ERCC1-XPF), and the mismatch repair complex, Msh2-Msh3, are required for Z-DNA-induced genetic instability in yeast and human cells. Both ERCC1-XPF and MSH2-MSH3 bind to Z-DNA-forming sequences, though ERCC1-XPF recruitment to Z-DNA is dependent on MSH2-MSH3. Moreover, ERCC1-XPF−dependent DNA strand-breaks occur near the Z-DNA-forming region in human cell extracts, and we model these interactions at the sub-molecular level. We propose a relationship in which these complexes recognize and process Z-DNA in eukaryotes, representing a mechanism of Z-DNA-induced genomic instability.

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.

Structure and mechanism of pyrimidine-pyrimidone (6-4) photoproduct recognition by the Rad4/XPC nucleotide excision repair complex.

Failure in repairing ultraviolet radiation-induced DNA damage can lead to mutations and cancer. Among UV-lesions, the pyrimidine–pyrimidone (6-4) photoproduct (6-4PP) is removed from the genome much faster than the cyclobutane pyrimidine dimer (CPD), owing to the more efficient recognition of 6-4PP by XPC-RAD23B, a key initiator of global-genome nucleotide excision repair (NER). Here, we report a crystal structure of a Rad4–Rad23 (yeast XPC-Rad23B ortholog) bound to 6-4PP-containing DNA and 4-μs molecular dynamics (MD) simulations examining the initial binding of Rad4 to 6-4PP or CPD. This first structure of Rad4/XPC bound to a physiological substrate with matched DNA sequence shows that Rad4 flips out both 6-4PP-containing nucleotide pairs, forming an ‘open’ conformation. The MD trajectories detail how Rad4/XPC initiates ‘opening’ 6-4PP: Rad4 initially engages BHD2 to bend/untwist DNA from the minor groove, leading to unstacking and extrusion of the 6-4PP:AA nucleotide pairs towards the major groove. The 5′ partner adenine first flips out and is captured by a BHD2/3 groove, while the 3′ adenine extrudes episodically, facilitating ensuing insertion of the BHD3 β-hairpin to open DNA as in the crystal structure. However, CPD resists such Rad4-induced structural distortions. Untwisting/bending from the minor groove may be a common way to interrogate DNA in NER.

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex.

Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions.

ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex

DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 "opens" up damaged DNA by inserting a β-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion "opening" is slow (˜5-10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-μs step that we assign to nonspecific deformation (unwinding/"twisting") of DNA by Rad4. The β-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise "twist-open" mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins.

UV induced ubiquitination of the yeast Rad4-Rad23 complex promotes survival by regulating cellular dNTP pools.

Regulating gene expression programmes is a central facet of the DNA damage response. The Dun1 kinase protein controls expression of many DNA damage induced genes, including the ribonucleotide reductase genes, which regulate cellular dNTP pools. Using a combination of gene expression profiling and chromatin immunoprecipitation, we demonstrate that in the absence of DNA damage the yeast Rad4–Rad23 nucleotide excision repair complex binds to the promoters of certain DNA damage response genes including DUN1, inhibiting their expression. UV radiation promotes the loss of occupancy of the Rad4–Rad23 complex from the regulatory regions of these genes, enabling their induction and thereby controlling the production of dNTPs. We demonstrate that this regulatory mechanism, which is dependent on the ubiquitination of Rad4 by the GG-NER E3 ligase, promotes UV survival in yeast cells. These results support an unanticipated regulatory mechanism that integrates ubiquitination of NER DNA repair factors with the regulation of the transcriptional response controlling dNTP production and cellular survival after UV damage.

Melanocyte UV resistance: Feelin' the endothelin.

Melanocyte UV resistance: Feelin' the endothelin.

Altered Expression and DNA Methylation Profiles of ERCC6 Gene in Lens Tissue from Age-Related Cortical Cataract

Ultraviolet (UV)-induced DNA damage attributes to the pathogenesis of age-related cataract (ARC) and is repaired via the nucleotide excision repair (NER). It is known that Cockayne syndrome complementation group B (CSB) protein coded by ERCC6 is a component of NER complex. DNA methylation is one of the major epigenetic events and is catalyzed by DNA 5-cytosine-methyltransferases (DNMTs). This study was to examine the potential contribution of DNA methylation of CpG islands in ERCC6 promoter region in lens tissues to ARC pathogenesis. Fifteen cortical type of ARC lenses and fifteen transparent lenses from human subjects were included in this study. ERCC6 and DNMTs expression in the lenses were analyzed by qRT-PCR and western blot. Bisulfite-sequencing PCR (BSP) was performed to evaluate methylation status of ERCC6. An in-vitro experiment by adding a demethylating agent 5-aza-2’-deoxycytidine (5-aza-dC) in Human lens epithelium B-3 (HLE B-3) was conducted to confirm the role of DNA methylation in ERCC6 expression. The results show that the mRNA and protein levels of ERCC6 were significantly reduced in the LECs and lens cortex of ARCs. DNMT3b mRNA was significantly higher in LECs of ARCs than that of the controls. In ARC group, the CpG island in the promoter region of ERCC6 displayed hypermethylation in LECs compared to that of the controls. After treatment with 5-aza-dC, the ERCC6 protein level increased in HLE B-3. We concluded that the overexpression of DNMT3b in lens is associated to the hypermethylation of the CpG island of ERCC6, which linked to the reduced ERCC6 expression in LECs from ARC patients. This epigenetic change in ERCC6 gene might be a factor of ARC formation that is mediated with impaired DNA repair.

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.

Structural and functional evidence that Rad4 competes with Rad2 for binding to the Tfb1 subunit of TFIIH in NER

XPC/Rad4 (human/yeast) recruits transcription faction IIH (TFIIH) to the nucleotide excision repair (NER) complex through interactions with its p62/Tfb1 and XPB/Ssl2 subunits. TFIIH then recruits XPG/Rad2 through interactions with similar subunits and the two repair factors appear to be mutually exclusive within the NER complex. Here, we show that Rad4 binds the PH domain of the Tfb1 (Tfb1PH) with high affinity. Structural characterization of a Rad4–Tfb1PH complex demonstrates that the Rad4-binding interface is formed using a motif similar to one used by Rad2 to bind Tfb1PH. In vivo studies in yeast demonstrate that the N-terminal Tfb1-binding motif and C-terminal TFIIH-binding motif of Rad4 are both crucial for survival following exposure to UV irradiation. Together, these results support the hypothesis that XPG/Rad2 displaces XPC/Rad4 from the repair complex in part through interactions with the Tfb1/p62 subunit of TFIIH. The Rad4–Tfb1PH structure also provides detailed information regarding, not only the interplay of TFIIH recruitment to the NER, but also links the role of TFIIH in NER and transcription.

Nucleotide Excision Repair Is Associated with the Replisome and Its Efficiency Depends on a Direct Interaction between XPA and PCNA

Proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP)-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM). The xeroderma pigmentosum group A (XPA) protein, with a central but somewhat elusive role in nucleotide excision repair (NER), contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA−/− cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6–4) photoproducts, and are consequently more arrested in S phase as compared to XPA−/− cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair.