Xeroderma Pigmentosum Complementation Group A Research Articles

XPA tumor variant leads to defects in NER that sensitize cells to cisplatin.

Nucleotide excision repair (NER) reduces efficacy of treatment with platinum (Pt)-based chemotherapy by removing Pt lesions from DNA. Previous study has identified that missense mutation or loss of the NER genes Excision Repair Cross Complementation Group 1 and 2 (ERCC1 and ERCC2) leads to improved patient outcomes after treatment with Pt-based chemotherapies. Although most NER gene alterations found in patient tumors are missense mutations, the impact of mutations in the remaining nearly 20 NER genes is unknown. Towards this goal, we previously developed a machine learning strategy to predict genetic variants in an essential NER protein, Xeroderma Pigmentosum Complementation Group A (XPA), that disrupt repair. In this study, we report in-depth analyses of a subset of the predicted variants, including in vitro analyses of purified recombinant protein and cell-based assays to test Pt agent sensitivity in cells and determine mechanisms of NER dysfunction. The most NER deficient variant Y148D had reduced protein stability, weaker DNA binding, disrupted recruitment to damage, and degradation. Our findings demonstrate that tumor mutations in XPA impact cell survival after cisplatin treatment and provide valuable mechanistic insights to improve variant effect prediction. Broadly, these findings suggest XPA tumor variants should be considered when predicting chemotherapy response.

An Active Learning Framework Improves Tumor Variant Interpretation.

A novel machine learning approach predicts the impact of tumor mutations on cellular phenotypes, overcomes limited training data, minimizes costly functional validation, and advances efforts to implement cancer precision medicine.

301 Comprehensive analyses on melanocytes differentiated from induced pruripotent stem cells originated from xeroderma pigmentosum complementation group A

Xeroderma Pigmentosum (XP) is a rare, autosomal recessive, hereditary DNA repair disorder characterized by extreme hypersensitivity to ultraviolet radiation (UVR). XP complementation group A (XP-A) is the most frequent type in Japan, and patients with XP-A present most severe cutaneous and neurological symptoms due to nucleotide excision repair deficiency. Here, we succeeded in generating melanocytes from induced pruripotent stem cells (iPSCs) derived from XP-A patients via melanocyte precursor cells (MPCs), and performed comprehensive analyses of gene expression to investigate the underlying molecular mechanisms of the disease. XP-A-iPSC- derived melanocytes (XP-A-iMCs) appeared morphologically similar to normal human epidermal melanocytes and expressed similarly melanocyte markers. We analyzed difference of the microarray data between XP-A-iMCs and healthy-control-iPSC-derived melanocytes (HC-iMCs) at 12 hours after high dose (150 J/m2) UV-B irradiation. Consequently we found that the major GO term categories for genes specifically upregulated in XP-A-iMCs were cell proliferation and death, and that for genes specifically downregulated in XP-A-iMCs was cell cycle regulation. These results suggest that XP-A-iMCs have increased apoptosis and cell proliferation compared to HC-iMCs. This phenotype of XP-A-iMCs may be responsible for the pigmented and depigmented maculae characteristic of XP-A patients.

XPA deficiency affects the ubiquitin-proteasome system function

Xeroderma pigmentosum complementation group A (XPA), is defective in xeroderma pigmentosum patients, causing pre-disposition to skin cancer and neurological abnormalities, which is not well understood. Here, we analyzed the XPA-deficient cells transcriptional profile under oxidative stress. The imbalance in of ubiquitin-proteasome system (UPS) gene expression was observed in XPA-deficient cells and the involvement of nuclear factor erythroid 2-related factor-2 (NFE2L2) was indicated. Co-immunoprecipitation assays showed the interaction between XPA, apurinic-apyrimidinic endonuclease 1 (APE1) and NFE2L2 proteins. Decreased NFE2L2 protein expression and proteasome activity was also observed in XPA-deficient cells. The data suggest the involvement of the growth arrest and DNA-damage-inducible beta (GADD45β) in NFE2L2 functions. Similar results were obtained in xpa-1 (RNAi) Caenorhabditis elegans suggesting the conservation of XPA and NFE2L2 interactions. In conclusion, stress response activation occurs in XPA-deficient cells under oxidative stress; however, these cells fail to activate the UPS cytoprotective response, which may contribute to XPA patient’s phenotypes.

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 β-hairpin. A conserved tryptophan residue Trp175 packs against the last base pair of DNA duplex and stabilizes the conformation of the characteristic β-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.

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 Å resolution. 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.

Nucleotide excision repair capacity increases during differentiation of human embryonic carcinoma cells into neurons and muscle cells

Embryonic stem cells can self-renew and differentiate, holding great promise for regenerative medicine. They also employ multiple mechanisms to preserve the integrity of their genomes. Nucleotide excision repair, a versatile repair mechanism, removes bulky DNA adducts from the genome. However, the dynamics of the capacity of nucleotide excision repair during stem cell differentiation remain unclear. Here, using immunoslot blot assay, we measured repair rates of UV-induced DNA damage during differentiation of human embryonic carcinoma (NTERA-2) cells into neurons and muscle cells. Our results revealed that the capacity of nucleotide excision repair increases as cell differentiation progresses. We also found that inhibition of the apoptotic signaling pathway has no effect on nucleotide excision repair capacity. Furthermore, RNA-Seq-based transcriptomic analysis indicated that expression levels of four core repair factors, xeroderma pigmentosum (XP) complementation group A (XPA), XPC, XPG, and XPF-ERCC1, are progressively up-regulated during differentiation, but not those of replication protein A (RPA) and transcription factor IIH (TFIIH). Together, our findings reveal that increase of nucleotide excision repair capacity accompanies cell differentiation, supported by the up-regulated transcription of genes encoding DNA repair enzymes during differentiation of two distinct cell lineages.

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 Å resolution, 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

Editor's Note: Prevention of Ultraviolet Radiation-Induced Immunosuppression by (-)-Epigallocatechin-3-Gallate in Mice Is Mediated through Interleukin 12-Dependent DNA Repair.

Purpose: Solar UV radiation–induced immunosuppression is considered to be a risk factor for melanoma and nonmelanoma skin cancers. We previously have shown that topical application of (−)-epigallocatechin-3-gallate (EGCG) prevents UV-induced immunosuppression in mice. We studied whether prevention of UV-induced immunosuppression by EGCG is mediated through interleukin 12 (IL-12)–dependent DNA repair. Experimental Design: IL-12 knockout (KO) mice on C3H/HeN background and DNA repair–deficient cells from xeroderma pigmentosum complementation group A ( XPA ) patients were used in this study. The effect of EGCG was determined on UV-induced suppression of contact hypersensitivity and UV-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in mice and XPA -deficient cells using immunohistochemistry and dot-blot analysis. Results: Topical treatment with EGCG prevented UV-induced suppression of the contact hypersensitivity in wild-type (WT) mice but did not prevent it in IL-12 KO mice. Injection of anti-IL-12 monoclonal antibody to WT mice blocked the preventive effect of EGCG on UV-induced immunosuppression. EGCG reduced or repaired UV-induced DNA damage in skin faster in WT mice as shown by reduced number of CPDs + cells and reduced the migration of CPD + antigen-presenting cells from the skin to draining lymph nodes. In contrast, this effect of EGCG was not seen in IL-12 KO mice. Further, EGCG was able to repair UV-induced CPDs in XPA -proficient cells obtained from healthy person but did not repair in XPA -deficient cells, indicating that nucleotide excision repair mechanism is involved in DNA repair. Conclusions: These data identify a new mechanism by which EGCG prevents UV-induced immunosuppression, and this may contribute to the chemopreventive activity of EGCG in prevention of photocarcinogenesis.

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.

Characterizing the Binding Interactions between DNA-Binding Proteins, XPA and XPE: A Molecular Dynamics Approach.

The scaffold nature of Xeroderma pigmentosum complementation group A (XPA) protein makes it an important member of nucleotide excision repair (NER) that removes bulky DNA lesions with the help of various protein–protein interactions (PPI) and DNA–protein interactions. However, many structural insights of XPA’s interaction and the binding patterns with other NER proteins are yet to be understood. Here, we have studied one such crucial PPI of XPA with another NER protein, Xeroderma pigmentosum complementation group A (XPE), by using the previously identified binding site of XPA (residues 185–226) in the Assisted Model Building With Energy Refinement force-field-mediated dynamic system. We studied the relationship between XPA185–226–XPE complex using three different docked models. The major residues observed in all of the models that were responsible for the PPI of this complex were Arg20, Arg47, Asp51, and Leu57 from XPE and the residues Leu191, Gln192, Val193, Trp194, Glu198, Glu202, Glu205, Arg207, Glu209, Gln216, and Phe219 from XPE185–226. During the simulation study, the orientation of XPA was also noted to be changed by almost 180° in models 1 and 3, which remain unchanged in model 2, indicating that XPA interacts with XPE with its N-terminal end facing downward and C-terminal end facing upward. The same was concurrent with the binding of DNA-binding domain region of XPA (aa98–239) with XPE. The N-terminal of XPE was stretched for accommodating XPA. Using the per-residue energy decomposition analysis for the interface residues of all models, the binding affinity between these proteins were found to be dependent on R20, R47, and L57 of XPE and the residues L191, V193, W194, E198, E202, E205, R207, and F219 of XPA. The net binding free energy of the XPA185–226–XPE protein complex was found to be −48.3718 kcal mol–1 for model 1, −49.09 kcal mol–1 for model 2, and −56.51 kcal mol–1 for model 3.

XPA expression is a predictive marker of the effectiveness of neoadjuvant chemotherapy for locally advanced uterine cervical cancer.

The standard treatment for locally advanced uterine cervical cancer is concurrent chemoradiotherapy. Successful neoadjuvant chemotherapy (NAC) may reduce tumor size and facilitate a hysterectomy, thereby improving the prognosis for patients with locally advanced cervical cancer. In contrast, unsuccessful NAC may worsen the prognosis because if a hysterectomy is not possible, the change in treatment plan may delay the initiation of core treatment. Therefore, there is a need to identify biomarkers that predict the efficacy of NAC in patients with uterine cervical cancer. The xeroderma pigmentosum complementation group A (XPA) protein serves a major role in nucleotide excision repair, which is a key DNA damage response pathway involved in cisplatin resistance. In the present study, the association between XPA expression in tumor tissue and the efficacy of NAC for locally advanced uterine cervical cancer was investigated. Data from 56 patients aged <70 years with locally advanced uterine cervical cancer (FIGO stages IIIA or IIIB) who were classified into two groups based on effective (n=31) and ineffective (n=25) responses to NAC treatment was evaluated. Tumor tissue samples were obtained by punch biopsy prior to NAC and XPA expression was examined immunohistochemically and scored using a weighted scoring system. In addition, the effects of RNA interference-mediated downregulation of XPA on the cisplatin sensitivity of uterine cervical cancer cells was investigated in vitro. It was revealed that the NAC effective group had significantly lower weighted XPA scores than the NAC ineffective group (P=0.001). Similarly, low tumor expression of XPA was significantly associated with higher sensitivity to NAC (P=0.001). Additionally, the downregulation of XPA expression in cervical cancer cells significantly increased their sensitivity to cisplatin in vitro. The results of the present study suggest that low XPA expression may be a predictive biomarker of NAC efficacy for patients with locally advanced uterine cervical cancer, which may be helpful for improving their prognosis.

In vivo Exposure Effects of 99mTc-methoxyisobutylisonitrile on the FDXR and XPA Genes Expression in Human Peripheral Blood Lymphocytes.

Objective(s):In recent years, the application of radiopharmaceuticals in nuclear medicine has increased substantially. Following the diagnostic procedures performed in nuclear medicine departments, such as myocardial perfusion imaging, patients generally receive considerable doses of radiation. Normally, radiation-induced DNA damages are expected following exposure to a low-dose ionizing radiation. In order to detect molecular changes, high-sensitivity techniques must be utilized. The aim of this study was to assess the effect of a low-dose (below 10 mSv) gamma ray on gene expression using quantitative real-time polymerase chain reaction (qRT-PCR).Methods:Blood samples were obtained from 20 volunteer patients who underwent myocardial perfusion imaging. They were given various doses of Technetium-99m methoxyisobutylisonitrile (99mTc-MIBI). After that, peripheral blood mononuclear cells (PBMNs) were derived, and then total RNA was extracted and reverse-transcribed to cDNA. Finally, the expression levels of xeroderma pigmentosum complementation group-A (XPA) and ferredoxin reductase (FDXR) genes were determinded through qRT-PCR technique using SYBR Green.Results:XPA and FDXR expression levels were obtained following a very low-dose ionizing radiation. A significant up-regulation of both genes was observed, and the gene expression level of each individual patient was different. If differences in the administered activity and radiosensitivity are taken into account, the observed differences could be justified. Furthermore, gender and age did not play a significant role in the expression levels of the genes under study.Conclusion:The up-regulation of FDXR after irradiation revealed the high-sensitivity level of this gene; therefore, it could be used as an appropriate biomarker for biological dosimetry. On the other hand, the up-regulation of XPA is an indication of DNA repair following radiation exposure. According to linear no-threshold model (LNT) and the results obtained from this study, a very low dose of ionizing radiation could bring about adverse biological effects at molecular level in the irradiated person.

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.

Brain atrophy and clinical severity in patients with Xeroderma Pigmentosum group A

Background: Xeroderma pigmentosum (XP) is an inherited congenital disease presenting with dermatological and neurological manifestations. In Japan, XP complementation group A (XP-A) is most frequently observed in eight clinical subtypes, suffer from severe manifestations including progressive brain atrophy since childhood.

NEIL1 protects against aflatoxin-induced hepatocellular carcinoma in mice

Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB1) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB1-induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB1-deoxyguanosine adduct (AFB1-Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB1 show significant increases in the levels of AFB1-Fapy-dG in Neil1-/- vs. wild-type liver DNA. Further, Neil1-/- mice are highly susceptible to AFB1-induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1-/- The magnitude of this effect in Neil1-/- mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB1-associated HCCs.

Regulation of the Functional Properties of Xeroderma Pigmentosum Complementation Group A (XPA) Protein through Lysine Acetylation

Nucleotide Excision Repair (NER) is the main repair pathway through which DNA damages occurring on account of ultraviolet (UV) or ionizing radiation (IR) are repaired. Xeroderma pigmentosum complementation group A (XPA) protein is a crucial part of the NER multi‐protein complex that plays an indispensable role in recognizing and binding the damaged DNA, as well as positioning other NER proteins optimally for efficient repair of the injury. Studies have shown that XPA can be dynamically acetylated and deacetylated impacting its involvement in the NER repair pathway. Deacetylation of XPA by SIRT1 enhances its interaction with the single strand DNA (ssDNA) binding protein, replication protein A (RPA). Our current research is focused on evaluating the effects of lysine acetylation on the structural and functional properties of XPA. Our mass spectrometry analysis revealed that XPA is in vitro acetylated at multiple sites by p300 and CBP acetyltransferases. We evaluated the effects of acetylation on the DNA binding properties of XPA protein using electrophoretic‐mobility shift assays (EMSA) and bio‐layer interferometry (BLI) technology. Interestingly, our results show that acetylation of XPA significantly lowers its binding affinity for cisplatin damaged DNA. Similar to reports in vivo, analysis of protein‐protein interactions between XPA and RPA, revealed that acetylation of XPA reduces its ability to interact with RPA. Our results suggest that acetylation of XPA negatively affects its role in recognizing, binding and consequently repairing damaged DNA, which in turn may impact subsequent recruitment of RPA. By application, our results present a mechanistic approach towards negatively modulating the effectiveness of the NER pathway, with the aim of improving the efficacy of current chemotherapeutics.Support or Funding InformationThis work was supported by a grant from the National Institutes of Health GM0983289 (to LB)

Hearing Dysfunction in Xpa-Deficient Mice.

Xeroderma pigmentosum (XP) is a rare recessive heredity disease caused by DNA repair impairment characterized by photosensitivity and neurologic symptoms in half of the cases. There are eight subtypes of XP: XP-A–XP-G and XP variant. Among eight subtypes, XP complementation group A (XP-A) display the lowest DNA repair ability and the severest cutaneous and neurologic symptoms. While its pathogenesis of skin symptoms have been well-studied, that of neurological symptoms, including sensorineural hearing loss (SNHL) remains unknown. Basic studies have suggested that SNHL may be caused by inner ear damage, including damage to the spiral ganglion neurons and organ of Corti, and that the XP-A is associated with most severe form of SNHL in humans. Here, we report the occurrence of SNHL in Xpa-deficient mice. Xpa-deficient mice and wild-type mice underwent measurements for auditory brainstem response, and the results revealed that Xpa-deficient mice exhibited significantly greater (p < 0.01) ABR thresholds at 4, 8, and 16 kHz than the wild-type mice. Furthermore, the number of spiral ganglion neurons was reduced in Xpa-deficient mice compared with that in wild-type mice, indicating that hearing loss may be related to spiral ganglion neuron deficiency, consistent with the few reports published in human patients with XP. These results provide important insights into the pathogenesis of SNHL in patients with XP-A.

Exopolysaccharides Isolated from Milk Fermented with Lactic Acid Bacteria Prevent Ultraviolet-Induced Skin Damage in Hairless Mice.

Background: We studied the mechanism by which fermented milk ameliorates UV-B-induced skin damage and determined the active components in milk fermented with lactic acid bacteria by evaluating erythema formation, dryness, epidermal proliferation, DNA damage and cytokine mRNA levels in hairless mice exposed to acute UV-B irradiation. Methods: Nine week-old hairless mice were given fermented milk (1.3 g/kg BW/day) or exopolysaccharide (EPS) concentrate (70 mg/kg BW/day) orally for ten days. Seven days after fermented milk or EPS administration began, the dorsal skin of the mice was exposed to a single dose of UV-B (20 mJ/cm2). Results: Ingestion of either fermented milk or EPS significantly attenuated UV-B-induced erythema formation, dryness and epidermal proliferation in mouse skin. Both fermented milk and EPS were associated with a significant decrease in cyclobutane pyrimidine dimers and upregulated mRNA levels of xeroderma pigmentosum complementation group A (XPA), which is involved in DNA repair. Furthermore, administration of either fermented milk or EPS significantly suppressed increases in the ratio of interleukin (IL)-10/IL-12a and IL-10/interferon-gamma mRNA levels. Conclusion: Together, these results indicate that EPS isolated from milk fermented with lactic acid bacteria enhanced DNA repair mechanisms and modulated skin immunity to protect skin against UV damage.