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 XPA_98–210 in DNA bound and DNA free state. Atomistic molecular dynamics simulation was carried out for both XPA_98–210 systems using AMBER force fields. We observed that XPA_98–210 in presence of damaged DNA exhibited more structural changes compared to XPA_98–210 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 β-sheets consisting mainly lysine residues.     

Modeling xeroderma pigmentosum associated neurological pathologies with patients-derived iPSCs

Xeroderma pigmentosum (XP) is a group of genetic disorders caused by mutations of XP-associated genes, resulting in impairment of DNA repair. XP patients frequently exhibit neurological degeneration, but the underlying mechanism is unknown, in part due to lack of proper disease models. Here, we generated patientspecific induced pluripotent stem cells (iPSCs) harboring mutations in five different XP genes including XPA, XPB, XPC, XPG, and XPV. These iPSCs were further differentiated to neural cells, and their susceptibility to DNA damage stress was investigated. Mutation of XPA in either neural stem cells (NSCs) or neurons resulted in severe DNA damage repair defects, and these neural cells with mutant XPA were hyper-sensitive to DNA damage-induced apoptosis. Thus, XP-mutant neural cells represent valuable tools to clarify the molecular mechanisms of neurological abnormalities in the XP patients.     

Effects of inhibitors on repair of DNA in normal human and xeroderma pigmentosum cells after exposure to x-rays and ultraviolet irradiation

Experiments were carried out to obtain direct evidence for the hypothesis that in human cells the repair of UV-damaged DNA is initiated by an incision step, and that this step is defective in cells from patients having Xeroderma pigmentosum (XP). The alkaline sucrose gradient centrifugation technique was used to detect breaks in the DNA.A decreased sedimentation velocity of the DNA was found after exposure of normal and XP cells to high doses of UV (5000 erg/mm²). Breaks were induced in the DNA by the UV irradiation without the action of an enzyme. After exposure of both types of cell to UV doses of 100–500 erg/mm², breaks that might occur by enzymic incision were not observed, possibly because of immediate rejoining.After single-strand breaks had been induced by X-rays, rejoining did not occur at temperatures lower than 22°. Rejoining was inhibited by KCN, 2,4-dinitrophenol, EDTA, iodoacetate and crystal violet. Actinomycin D, acriflavine and phleomycin, also tested as potential inhibitors of the repair process, induced breaks or conformational changes in the DNA of unirradiated normal and XP cells.Application to UV-exposed cells of conditions that inhibit the rejoining of breaks did not cause accumulation of breaks in the DNA. The results suggest a coordinated and sequential performance of the steps in the repair of each UV lesion by repair enzymes which may act as a complex.     

Replication of damaged DNA: molecular defect in xeroderma pigmentosum variant cells.

Individuals with Xeroderma pigmentosum (XP) syndrome have a genetic predisposition to sunlight-induced skin cancer. Genetically different forms of XP have been identified by cell fusion. Cells of individuals expressing the classical form of XP (complementation groups A through G) are deficient in the nucleotide excision repair (NER) pathway. In contrast, the cells belonging to the variant class of XP (XPV) are NER-proficient and are only slightly more sensitive than normal cells to the killing action of UV light radiation. The XPV fibroblasts replicate damaged DNA generating abnormally short fragments either in vivo [A.R. Lehmann, The relationship between pyramidine dimers and replicating DNA in UV-irradiated human fibroblasts, Nucleic Acids Res. 7 (1979) 1901-1912; S.D. Park, J.E. Cleaver, Postreplication repair: question of its definition and possible alteration in Xeroderma pigmentosum cell strains, Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 3927-3931.] or in vitro [S.M. Cordeiro, L.S. Zaritskaya, L.K. Price, W.K. Kaufmann, Replication fork bypass of a pyramidine dimer blocking leading strand DNA synthesis, J. Biol. Chem. 272 (1997) 13945-13954; D.L. Svoboda, L.P. Briley, J.M. Vos, Defective bypass replication of a leading strand cyclobutane thymine dimer in Xeroderma pigmentosum variant cell extracts, Cancer Res. 58 (1998) 2445-2448; I. Ensch-Simon, P.M. Burgers, J.S. Taylor, Bypass of a site-specific cis-syn thymine dimer in an SV40 vector during in vitro replication by HeLa and XPV cell-free extracts, Biochemistry 37 (1998) 8218-8226.], suggesting that in XPV cells, replication has an increased probability of being blocked at a lesion. Furthermore, extracts from XPV cells were found to be defective in translesion synthesis [A. Cordonnier, A.R. Lehmann, R.P.P. Fuchs, Impaired translesion synthesis in Xeroderma pigmentosum variant extracts, Mol. Cell. Biol. 19 (1999) 2206-2211.]. Recently, Masutani et al. [C. Masutani, M. Araki, A. Yamada, R. Kusomoto, T. Nogimori, T. Maekawa, S. Iwai, F. Hanaoka, Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity, EMBO J. 18 (1999) 3491-3501.] have shown that the XPV defect can be corrected by a novel human DNA polymerase, homologue to the yeast DNA polymerase eta, which is able to replicate past cyclobutane pyrimidine dimers in DNA templates. This review focuses on our current understanding of translesion synthesis in mammalian cells whose defect, unexpectedly, is responsible for the hypermutability of XPV cells and for the XPV pathology.     

Contribution of defective mitophagy to the neurodegeneration in DNA repair-deficient disorders

DNA repair is a prerequisite for life as we know it, and defects in DNA repair lead to accelerated aging. Xeroderma pigmentosum group A (XPA) is a classic DNA repair-deficient disorder with patients displaying sun sensitivity and cancer susceptibility. XPA patients also exhibit neurodegeneration, leading to cerebellar atrophy, neuropathy, and hearing loss, through a mechanism that has remained elusive. Using in silico, in vitro, and in vivo studies, we discovered defective mitophagy in XPA due to PARP1 hyperactivation and NAD⁺ (and thus, SIRT1) depletion. This leads to mitochondrial membrane hyper-polarization, PINK1 cleavage and defective mitophagy. This study underscores the importance of mitophagy in promoting a healthy pool of mitochondria and in preventing neurodegeneration and premature aging.     

Contribution of defective mitophagy to the neurodegeneration in DNA repair-deficient disorders

DNA repair is a prerequisite for life as we know it, and defects in DNA repair lead to accelerated aging. Xeroderma pigmentosum group A (XPA) is a classic DNA repair-deficient disorder with patients displaying sun sensitivity and cancer susceptibility. XPA patients also exhibit neurodegeneration, leading to cerebellar atrophy, neuropathy, and hearing loss, through a mechanism that has remained elusive. Using in silico, in vitro, and in vivo studies, we discovered defective mitophagy in XPA due to PARP1 hyperactivation and NAD⁺ (and thus, SIRT1) depletion. This leads to mitochondrial membrane hyper-polarization, PINK1 cleavage and defective mitophagy. This study underscores the importance of mitophagy in promoting a healthy pool of mitochondria and in preventing neurodegeneration and premature aging.     

Adenovirus mediated transduction of the human DNA polymerase eta cDNA

Xeroderma pigmentosum (XP) is an autosomal recessive photosensitive disorder with an extremely high incidence of skin cancers. Seven complementation groups, corresponding to seven proteins involved in nucleotide excision repair (NER), are associated with this syndrome. However, in XP variant patients, the disorder is caused by defects in DNA polymerase eta; this error prone polymerase, encoded by POLH, is involved in translesion DNA synthesis (TLS) on DNA templates damaged by ultraviolet light (UV). We constructed a recombinant adenovirus carrying the human POLH cDNA linked to the EGFP reporter gene (AdXPV-EGFP) and infected skin fibroblasts from both XPV and XPA patients. Twenty-four hours after infection, the DNA polymerase eta-EGFP fusion protein was detected by Western blot analysis, demonstrating successful transduction by the adenoviral vector. Protein expression was accompanied by reduction in the high sensitivity of XPV cells to UV, as determined by cell survival and apoptosis-induction assays. Moreover, the pronounced UV-induced inhibition of DNA synthesis in XPV cells and their arrest in S phase were attenuated in AdXPV-EGFP infected cells, confirming that the transduced polymerase was functional. However, over-expression of polymerase eta mediated by AdXPV-EGFP infection did not result in enhancement of cell survival, prevention of apoptosis, or higher rate of nascent DNA strand growth in irradiated XPA cells. These results suggest that TLS by DNA polymerase eta is not a limiting factor for recovery from cellular responses induced by UV in excision-repair deficient fibroblasts.     

Use of UV endonuclease from Micrococcus luteus to monitor the progress of DNA repair in UV-irradiated human cells

A sensitive enzymatic assay has been developed to follow the progress of NDA repair in human cells exposed to UV radiation. The assay employs an endonuclease selectively active at sites containing pyrimidine dimers in UV-damaged DNA. Primary fibroblasts are exposed to 254 nm radiation and incubated for specified times, their radioactivity labelled DNA is isolated and treated with a UV endonuclease extensively purified from Micrococcus luteus. Endonuclease-susceptible site remaining in the DNA are subsequently observed as single-strand scissions by sedimentation in alkaline sucrose gradients. In comparison to the situation with excision-proficient normal cells, those derived from patients suffering from either the classical or the De Sanctis-Cacchione clinical form of Xeroderma pigmentosum (XP) exhibit a marked diminution in the rate of disappearance of nuclease-susceptible lesions with time of post-UV incubation.     

Xeroderma pigmentosum complementation group A protein is driven to nucleotide excision repair sites by the electrostatic potential of distorted DNA

The presumed DNA-binding cleft of xeroderma pigmentosum group A (XPA) protein, a key regulatory subunit of the eukaryotic nucleotide excision repair complex, displays a distinctive array of 6 positively charged amino acid side chains. Here, the molecular function of these closely spaced electropositive residues has been tested by systematic site-directed mutagenesis. After the introduction of single amino acid substitutions, the mutants were probed for protein-DNA interactions in electrophoretic mobility shift and photochemical crosslinking assays. This analysis led to the identification of a critical hot-spot for DNA substrate recognition composed of two neighboring lysines at codons 141 and 179 of the human XPA sequence. The replacement of other basic side chains in the DNA interaction domain conferred more moderate defects of substrate binding. When the function of XPA was tested as a fusion product with either mCherry or green-fluorescent protein, a glutamate substitution of one of the positively charged residues at positions 141 and 179 was sufficient to decrease DNA repair activity in human fibroblasts. Thus, the removal of a single cationic side chain abolished DNA-binding activity and significant excision repair defects could be induced by single charge inversions on the XPA surface, indicating that this molecular sensor participates in substrate recognition by monitoring the electrostatic potential of distorted DNA repair sites.     

Reactivation of UV and γ-inactivated herpes virus in BHK cells

The DNA-repair capabilities of baby hamster kidney (BHK) cells were investigated by comparing the reactivation of irradiated herpes simplex virus type I (HSV1) in BHK cells with its reactivation in mouse fibroblasts and in normal and repairdeficient human diploid fibroblasts. BHK cells were found to have an intermediate ability to reactive UV-irradiated HSV1 (the viral D_o was 14 J/m²) relative to normal human fibroblasts (viral D_o = 19 J/m²) and xeroderma pigmentosum (XP) group A cells (viral D_o = 4.5 J/m²). With mouse L929 cells as the host, the response of the UV-irradiated virus was biphasic with D_os of 4.6 and 30 J/m² for the low- and high-dose components respectively. In contrast to the response following UV radiation, γ-irradiated HSV1 was similarly reactivated by BHK and normal human cells (the D_os for the irradiated virus in BHK and CRl 1106 were 55 and 51 krad, respectively, whereas xeroderma pigmentosum cells were slightly less efficient in the repair of γ-irradiated virus (D_o = 45 krad). UV irradiation of BHK host cells 0–48 h prior to infection enhanced the reactivation of UV-irradiated HSV.     

Cytological evidence for DNA chain elongation after UV irradiation in the S phase

Human cells irradiated with UV light synthesize lower molecular weight DNA than unirradiated cells. This reduction in molecular weight is greater in xeroderma pigmentosum (XP) cells than in normal cells. The molecular weight of DNA is further reduced by the addition of caffeine to XP cells. By several hours after irradiation, DNA fragments are barely detectable. Cells from excision-proficient and excision-deficient XP patients were studied autoradiographically to produce cytological evidence of DNA chain elongation. Replicate cultures with and without caffeine were synchronized and irradiated with UV light during the S phase. Caffeine was removed in G₂, and the cells were labeled with ³H-thymidine. Results showed significantly increased labeling during G₂ of excision-deficient XP cells. Labeling was dependent on both time of irradiation and presence of caffeine. The XP variant cells had no increase in labeling for any irradiation time.Published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 1608. Supported by N.I.C. Grant TO1CA05170-10.     

Formation of DNA strand breaks in UV-irradiated human fibroblast preparations

The formation of DNA strand breaks was characterized in human fibroblasts prepared by several methods. In quiescent monolayer cultures of normal human fibroblasts (NHF), exposure to 254 nm radiation (UV) caused the rapid appearance of DNA strand breaks as monitored by alkaline elution analysis. Maximal levels of DNA breaks were seen 30 min after 10 J/m2; thereafter, strand breaks disappeared. Breakage soon after irradiation appeared to saturate at fluences above 10 J/m2. Xeroderma pigmentosum fibroblasts belonging to complementation group A (XPA) did not display this response which reflects operations of the nucleotidyl DNA excision repair pathway. When fibroblast strains were released from culture dishes by enzymatic digestion with trypsin or by scraping with a rubber policeman, UV-dependent DNA breakage displayed altered dose and time responses. Few breaks were detected in detached preparations of NHF after 10 J/m2 indicating inactivation of nucleotidyl DNA excision repair. The fluence response in detached fibroblasts was linear up to an incident fluence of 100 J/m2. Moreover, after 25 or 50 J/m2, strand breaks accumulated as a linear function of time for up to 2 h after irradiation. This UV-dependent and time-dependent incision activity was also observed in XPA monolayers and released-cell preparations. In permeable fibroblast preparations, DNA breaks accumulated in unirradiated cells that had been released with trypsin or by scraping. Permeabilization in situ saponin to open the plasma membrane produced a cell preparation that accumulated fewer UV-independent breaks. In saponin-permeabilized NHF that were irradiated with 10 J/m2, UV-dependent strand incision activity occurred at about 30% of the rate of incision seen in intact monolayer NHF. These results reveal at least 3 DNA strand incision activities in human fibroblast preparations of which only one reflects operation of the nucleotidyl DNA excision repair pathway.     

Defective repair of mitomycin C crosslinks in Fanconi's anemia and loss in confluent normal human and xeroderma pigmentosum cells

Crosslink repair of mitomycin C-induced interstrand crosslinks was studied in exponentially growing and confluent normal human, transformed WI38CT-1, Fanconi's anemia (FA) and xeroderma pigmentosum (XP) group-A fibroblasts by the assay methods of alkaline sucrose centrifugation, hydroxyapatite column chromatography and S₁-nuclease digestion. These three methods demonstrated unequivocally that crosslinking occurred at a rate of 0.13 crosslinks/10⁸ Da per μg per ml mitomycin C ( 10 μg/ml) and the first half-excision of crosslinks followed the rapid first-order kinetics of 2–3 h half-life in exponentially-growing normal, WI38CT-1 and XP group-A cells. However, the first half-excision was completely defective in three out of the four FA strains tested and severely retarded in an FA strain. These results strongly support our previous observations in different strains of normal human, FA and XP group-A cells. An important new addition is that confluent, otherwise proficient, normal and XP cells almost completely lost the ability of the first, rapid half-excision of mitomycin C crosslinks in their DNA. This probably suggests that the enzyme or regulatory factor responsible for the half-excision, which differs from that for nucleotide excision repair, present constitutively in confluent cells, may be induced or activated only in the cycling cells. However, its relation to a defective FA factor is not clear at present.     

High levels of oxidatively generated DNA damage 8,5′-cyclo-2′-deoxyadenosine accumulate in the brain tissues of xeroderma pigmentosum group A gene-knockout mice

Xeroderma pigmentosum (XP) is a genetic disorder associated with defects in nucleotide excision repair, a pathway that eliminates a wide variety of helix-distorting DNA lesions, including ultraviolet-induced pyrimidine dimers. In addition to skin diseases in sun-exposed areas, approximately 25% of XP patients develop progressive neurological disease, which has been hypothesized to be associated with the accumulation of an oxidatively generated type of DNA damage called purine 8,5′-cyclo-2′-deoxynucleoside (cyclopurine). However, that hypothesis has not been verified. In this study, we tested that hypothesis by using the XP group A gene-knockout (Xpa^−/−) mouse model. To quantify cyclopurine lesions in this model, we previously established an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (CdA-1) that specifically recognizes 8,5′-cyclo-2′-deoxyadenosine (cyclo-dA). By optimizing conditions, we increased the ELISA sensitivity to a detection limit of ˜one cyclo-dA lesion/10⁶ nucleosides. The improved ELISA revealed that cyclo-dA lesions accumulate with age in the brain tissues of Xpa^−/− and of wild-type (wt) mice, but there were significantly more cyclo-dA lesions in Xpa^−/− mice than in wt mice at 6, 24 and 29 months of age. These findings are consistent with the long-standing hypothesis that the age-dependent accumulation of endogenous cyclopurine lesions in the brain may be critical for XP neurological abnormalities.     

Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts

The inhibition of DNA replication in ultraviolet-irradiated human fibroblasts was characterized by quantitative analysis of radiation-induced alterations in the steady-state distribution of sizes of pulse-labeled, nascent DNA. Low, noncytotoxic fluences (<1 J/m², producing less than one pyrimidine dimer per replicon) rapidly produced an inhibition of DNA synthesis in half-replicon-size replication intermediates without noticeably affecting synthesis in multi-repliconsize intermediates. With time, the inhibition produced by low fluences spread progressively to include multi-replicon-size intermediates. The results indicate that ultraviolet radiation inhibits the initiation of DNA synthesis in replicons. Higher (>1 J/m², producing more than one dimer per replicon) cytotoxic fluences inhibited DNA synthesis in operating replicons presumably because the elongation of nascent strands was blocked where pyrimidine dimers were present in template strands. Xeroderma pigmentosum fibroblasts with deficiencies in DNA excision repair exhibited an inhibition of replicon initiation after low radiation fluences. indicating the effect was not solely dependent upon operation of the nucleotidyl excision repair pathway. Owing to their inability to remove pyrimidine dimers ahead of DNA growing points, the repair-deficient cells also were more sensitive than normal cells to the ultraviolet-induced inhibition of chain elongation. Xeroderma pigmentosum cells belonging to the variant class were even more sensitive to inhibition of chain elongation than the repair-deficient strains despite their ability to remove pyrimidine dimers. This analysis suggests that normal and repair-deficient human fibroblasts either are able to rapidly bypass certain dimers or these dimers are not recognized by the chain elongation machinery.     

Impaired translesion synthesis in xeroderma pigmentosum variant extracts

Xeroderma pigmentosum variant (XPV) cells are characterized by a cellular defect in the ability to synthesize intact daughter DNA strands on damaged templates. Molecular mechanisms that facilitate replication fork progression on damaged DNA in normal cells are not well defined. In this study, we used single-stranded plasmid molecules containing a single N-2-acetylaminofluorene (AAF) adduct to analyze translesion synthesis (TLS) catalyzed by extracts of either normal or XPV primary skin fibroblasts. In one of the substrates, the single AAF adduct was located at the 3' end of a run of three guanines that was previously shown to induce deletion of one G by a slippage mechanism. Primer extension reactions performed by normal cellular extracts from four different individuals produced the same distinct pattern of TLS, with over 80% of the products resulting from the elongation of a slipped intermediate and the remaining 20% resulting from a nonslipped intermediate. In contrast, with cellular extracts from five different XPV patients, the TLS reaction was strongly reduced, yielding only low amounts of TLS via the nonslipped intermediate. With our second substrate, in which the AAF adduct was located at the first G in the run, thus preventing slippage from occurring, we confirmed that normal extracts were able to perform TLS 10-fold more efficiently than XPV extracts. These data demonstrate unequivocally that the defect in XPV cells resides in translesion synthesis independently of the slippage process.     

Semiautomated autoradiographic measurement of DNA repair in normal and xeroderma pigmentosum cultured human fibroblasts

Summary Assessment of DNA repair in cultured human fibroblasts by autoradiography may be facilitated by using semiautomated grain counting instruments. The instrument-determined number of autoradiographic grains per nucleus in cultured human skin fibroblasts was found to be linear in comparison to visual counts up to only 30 grains per nucleus. However, with two different instruments a greater range of linearity (to 100 to 120 grains per nucleus) was attained by measuring the grain surface area per nucleus. Semiautomated analysis of the grain surface area per nucleus yielded measurements of relative rates of unscheduled DNA synthesis after ultraviolet irradiation in xeroderma pigmentosum and normal human fibroblasts, which were reproducible and rapid.     

In silico characterization of a novel pathogenic deletion mutation identified in XPA gene in a Pakistani family with severe xeroderma pigmentosum

Background

Xeroderma Pigmentosum (XP) is a rare skin disorder characterized by skin hypersensitivity to sunlight and abnormal pigmentation. The aim of this study was to investigate the genetic cause of a severe XP phenotype in a consanguineous Pakistani family and in silico characterization of any identified disease-associated mutation.

Results

The XP complementation group was assigned by genotyping of family for known XP loci. Genotyping data mapped the family to complementation group A locus, involving XPA gene. Mutation analysis of the candidate XP gene by DNA sequencing revealed a novel deletion mutation (c.654del A) in exon 5 of XPA gene. The c.654del A, causes frameshift, which pre-maturely terminates protein and result into a truncated product of 222 amino acid (aa) residues instead of 273 (p.Lys218AsnfsX5). In silico tools were applied to study the likelihood of changes in structural motifs and thus interaction of mutated protein with binding partners. In silico analysis of mutant protein sequence, predicted to affect the aa residue which attains coiled coil structure. The coiled coil structure has an important role in key cellular interactions, especially with DNA damage-binding protein 2 (DDB2), which has important role in DDB-mediated nucleotide excision repair (NER) system.

Conclusions

Our findings support the fact of genetic and clinical heterogeneity in XP. The study also predicts the critical role of DDB2 binding region of XPA protein in NER pathway and opens an avenue for further research to study the functional role of the mutated protein domain.