Abstract Semenenko, V. A., Stewart, R. D. and Ackerman, E. J. Monte Carlo Simulation of Base and Nucleotide Excision Repair of Clustered DNA Damage Sites. I. Model Properties and Predicted Trends. Radiat. Res. 164, 180–193 (2005). DNA is constantly damaged through endogenous processes and by exogenous agents, such as ionizing radiation. Base excision repair (BER) and nucleotide excision repair (NER) help maintain the stability of the genome by removing many different types of DNA damage. We present a Monte Carlo excision repair (MCER) model that simulates key steps in the short-patch and long-patch BER pathways and the NER pathway. The repair of both single and clustered damages, except double-strand breaks (DSBs), is simulated in the MCER model. Output from the model includes estimates of the probability that a cluster is repaired correctly, the fraction of the clusters converted into DSBs through the action of excision repair enzymes, the fraction of the clusters repaired with mutations, and the expected number of repair cycles needed to completely remove a clustered damage site. The quantitative implications of alternative hypotheses regarding the postulated repair mechanisms are investigated through a series of parameter sensitivity studies. These sensitivity studies are also used to help define the putative repair characteristics of clustered damage sites other than DSBs.

Confocal fluorescence optical microscopy and magnetic resonance microscopy are each used to study live cells in a minimally invasive way. Both techniques provide complementary information. Therefore, by examining cells simultaneously with both methodologies, more detailed information is obtained than is possible with each microscope individually. In this paper two configurations of a combined confocal and magnetic resonance microscope are described. The first configuration is capable of studying large single cells or three-dimensional cell agglomerates, whereas the second configuration is designed for the investigation of monolayers of mammalian cells. In both cases the sample compartment is part of a temperature regulated perfusion system. Images obtained with the combined system are shown forXenopus laevis oocytes, model JB6 tumor spheroids, and a single layer of Chinese hamster ovary cells. Finally, potential applications of the combined microscope are discussed.

Complementary data acquired with different microscopy techniques provide a basis for establishing a more comprehensive understanding of cell function in health and disease, particularly when results acquired with different methodologies can be correlated in time and space. In this article, a novel microscope is described for studying live cells simultaneously with both confocal scanning laser fluorescence optical microscopy and magnetic resonance microscopy. The various design considerations necessary for integrating these two complementary techniques are discussed, the layout and specifications of the instrument are given, and examples of confocal and magnetic resonance images of large frog cells and model tumor spheroids obtained with the compound microscope are presented.

Complementary data acquired with different microscopy techniques provide a basis for establishing a more comprehensive understanding of health and disease at a cellular level, particularly when data acquired with different methodologies can be correlated in both time and space. In this Communication, a brief description of a novel instrument capable of simultaneously performing confocal optical and magnetic resonance microscopy is presented, and the first combined images of live Xenopus laevis oocytes are shown. Also, the potential benefits of combined microscopy are discussed, and it is shown that the a priori knowledge of the high-resolution optical images can be used to enhance the boundary resolution and contrast of the MR images.

Nucleotide excision repair (NER) is a crucial pathway in the maintenance of genome stability requiring at least two dozen proteins. XPA and RPA have essential roles in the damage recognition step of NER. To better understand the mechanism of their interactions with DNA, we utilized equilibrium and stop-flow kinetic approaches with fluorescently labeled oligonucleotides. Fluorescein is a bona fide NER lesion because a circular plasmid with a single defined fluorescein was repaired by efficient extracts from Xenopus oocyte nuclei. Single-stranded and double-stranded oligonucleotides 5'-labeled with fluorescein were used in the subsequent studies. Oligonucleotide fluorescence was quenched upon specific binding to full-length recombinant Xenopus XPA (xXPA) and/or human RPA. The binding was highly sensitive to the buffer conditions. Analysis of equilibrium binding data with ds DNA and xXPA revealed a single dissociation constant (K-d) of 24.4 nM. Stopped-flow kinetic experiments were described by a first-order on-rate constant k(on) of 9.03 x 10(8) M-1 s(-1) and k(off) of 26.1 s(-1). From the ratio of off-rate to on-rate, a calculated K-d of 28.9 nM was obtained, revealing that the kinetic and equilibrium studies were consistent. The affinity of xXPA for ds undamaged DNA determined in our spectrofluorometry experiments was up to 3 orders of magnitude higher than previously reported values using different substrates, conditions, and assays [gel-shifts (EMSA), filter-binding, anisotropy, and surface plasmon resonance]. The same substrate DNA containing a 4-bp mismatch in the middle yielded a K-d five times higher (158 nM), indicating weaker binding by xXPA. The differences in Kd values for these two substrates were mainly attributable to the k(on), rather than k(off) rates. Fluorescence intensity changes upon interaction of xXPA with ss 50-mer were too low to calculate an accurate K-d. Although recombinant human RPA binding to the ds 50-mer was very weak (K-d > 1 mM), stop-flow and equilibrium measurements to ss oligonucleotide yielded K-d values of 96 and 20.3 nM, respectively, which correlated with previously reported values using gel mobility shift assays and a similarly sized poly-dT. Equilibrium and stop-flow measurements to the cognate and mismatched ds oligonucleotides using both xXPA and hRPA yielded a 2- to 3-fold increase in the K-d.

The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full-length protein, the minimal DNA-binding domain, revealed that one-third of this molecule was disordered. To better characterize structural features of full-length XPA, we performed time-resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS-PAGE. The molecular weight of the full-length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS-PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N- and C-terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well-characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85-I179) despite 14 possible cut sites. For the full-length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.

Eukaryotic DNA repair enzymes must interact with the architectural hierarchy of chromatin. The challenge of finding damaged DNA complexed with histone proteins in nucleosomes is complicated by the need to maintain local chromatin structures involved in regulating other DNA processing events. The heterogeneity of lesions induced by DNA-damaging agents has led us to design homogeneously damaged substrates to directly compare repair of naked DNA with that of nucleosomes. Here we report that nucleotide excision repair in Xenopus nuclear extracts can effectively repair a single UV radiation photoproduct located 5 bases from the dyad center of a positioned nucleosome, although the nucleosome is repaired at about half the rate at which the naked DNA fragment is. Extract repair within the nucleosome is >50-fold more rapid than either enzymatic photoreversal or endonuclease cleavage of the lesion in vitro. Furthermore, nucleosome formation occurs (after repair) only on damaged naked DNA (165-bp fragments) during a 1-h incubation in these extracts, even in the presence of a large excess of undamaged DNA. This is an example of selective nucleosome assembly by Xenopus nuclear extracts on a short linear DNA fragment containing a DNA lesion.

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTSingle-Molecule Conformational Dynamics of Fluctuating Noncovalent DNA−Protein Interactions in DNA Damage RecognitionH. Peter Lu, Lilia M. Iakoucheva, and Eric J. AckermanView Author Information Environmental Molecular Sciences Laboratory and Molecular Biosciences Department Pacific Northwest National Laboratory P.O. Box 999, Richland, Washington 99352 Cite this: J. Am. Chem. Soc. 2001, 123, 37, 9184–9185Publication Date (Web):August 22, 2001Publication History Received15 December 2000Revised18 July 2001Published online22 August 2001Published inissue 1 September 2001https://doi.org/10.1021/ja0058942Copyright © 2001 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views409Altmetric-Citations38LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (52 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Computational chemistry,Conformational transitions,Fluorescence,Genetics,Radioactive decay Get e-Alerts

The DNA repair protein XPA recognizes a wide variety of bulky lesions and interacts with several other proteins during nucleotide excision repair. We recently identified regions of intrinsic order and disorder in full length Xenopus XPA (xXPA) protein using an experimental approach that combined time‐resolved trypsin proteolysis and electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance (ESI‐FTICR) mass spectrometry (MS). MS data were consistent with the interpretation that xXPA contains no post‐translational modifications. Here we characterize the discrepancy between the calculated molecular weight (31 kDa) for xXPA and its apparent molecular weight on SDS‐PAGE (multiple bands from ∼40–45 kDa) and gel filtration chromatography (∼92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS‐PAGE yielded a single 42‐kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intramolecular glutaraldehyde crosslinks in xXPA produced a ∼32‐kDa monomer in closer agreement with its calculated molecular weight. To investigate whether binding to DNA alters xXPA's anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA ± cisplatin ± mismatches. xXPA showed at least 10‐fold higher affinity for cisplatin DNA ± mismatches compared to undamaged DNA ± mismatches. In all cases, DNA binding did not correct xXPA's anomalous migration. To test predictions that a Glu‐rich region (EEEEAEE) and/or disordered N‐ and C‐terminal domains were responsible for xXPA's aberrant mobility, the molecular weights of partial proteolytic fragments from ∼5 to 25 kDa separated by reverse‐phase HPLC and precisely determined by ESI‐FTICR MS were correlated with their migration on SDS‐PAGE. Every partial tryptic fragment analyzed within this size range exhibited 10%–50% larger molecular weights than expected. Thus, both the disordered domains and the Glu‐rich region in xXPA are primarily responsible for the aberrant mobility phenomena.

Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cell's capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.

Limited nucleotide excision repair (NER) requires at least ∼40 proteins in extracts from purified proteins (1,2) although perhaps hundreds of proteins may influence DNA repair in cells. For efficient DNA repair in extracts, it is important to utilize a system containing large quantities of active DNA repair proteins uncontaminated with nonspecific nucleases. Unlike extracts derived from mammalian cells that repair ∼2% of the input DNA, both injected Xenopus oocytes (3) and oocyte nuclear extracts can repair ∼100% of the input damaged DNA by NER with little or no synthesis on undamaged control substrate (Fig. 1). Repair activity in extracts can be inactivated with antibodies and/or inhibitors, and then repair can be restored by addition of exogenous proteins (4). A further advantage of the Xenopus system is that results obtained from injection experiments in living cells can be compared to results obtained in nuclear extracts.

UV‐induced photoproducts (cyclobutane pyrimidine dimers, CPDs) in DNA are removed by nucleotide excision repair (NER), and the presence of transcription factors on DNA can restrict the accessibility of NER enzymes. We have investigatigated the modulation of NER in a gene promoter using the Xenopus transcription factor IIIA (TFIIIA)–5S rDNA complex and Xenopus oocyte nuclear extracts. TFIIIA alters CPD formation primarily in the transcribed strand of the 50 bp internal control region (ICR) of 5S rDNA. During NER in vitro, CPD removal is reduced at most sites in both strands of the ICR when TFIIIA is bound. Efficient repair occurs just outside the TFIIIA‐binding site (within 10 bp), and in the absence of 5S rRNA transcription. Interestingly, three CPD sites within the ICR [+56, +75 (transcribed strand) and +73 (non‐transcribed strand)] are repaired rapidly when TFIIIA is bound, while CPDs within ∼5 bases of these sites are repaired much more slowly. CPDs at these three sites may partially displace TFIIIA, thereby enabling rapid repair. However, TFIIIA is not completely displaced during NER, at least at sites outside the ICR, even though the NER complex could be sterically hindered by TFIIIA. Such inefficient repair of transcription factor binding sites could increase mutation frequency in regulatory regions of genes.

Nucleotide excision repair (NER) is an important cellular mechanism, conserved from bacteria to humans, responsible for eliminating multiple types of structurally distinct DNA lesions from the genome. The protein XPA appears to play a central role in NER, recognizing and/or verifying damaged DNA and recruiting other proteins, including RPA, ERCC1, and TFIIH, to repair the damage. Sequence analysis and genetic evidence suggest that zinc, which is essential for DNA binding, is associated with a C4-type motif, C-X2-C-X17-C-X2-C. Sequence analysis suggests that a second, H2C2-type zinc-binding motif may be present near the C-terminal. Seventy percent of the amino acid sequence of Xenopus laevis XPA (xXPA) is identical to human XPA and both putative zinc-binding motifs are conserved in all known XPA proteins. Electrospray ionization-mass spectroscopy data show that xXPA contains only one zinc atom per molecule. EXAFS spectra collected on full-length xXPA in frozen (77 K) 15% glycerol aqueous solution unequivocally show that the zinc atom is coordinated to four sulfur atoms with an average Zn--S bond length of 2.33 +/- 0.02 A. Together, the EXAFS and mass spectroscopy data indicate that xXPA contains just one C4-type zinc-binding motif.

Xenopus oocytes and an oocyte nuclear extract efficiently repair the bulky DNA lesions cyclobutane pyrimidine dimers, (6-4) photoproducts, and N-acetoxy-2-aminofluorene (AAF) adducts by san excision repair mechanism. Nearly all (>95%) of the input damaged DNA was repaired within 5 h in both infected cells and extracts with no significant incorporation of label into control undamaged DNA. Remarkably, more than 10(10) cyclobutane pyrimidine dimers or (6-4) photoproducts are repaired/nuclei. The extracts are free from nuclease activity, and repair is independent of exogenous light. Both the high efficiency and DNA polymerase requirements of this system appear to be different from extracts derived from human cells. We demonstrated a requirement for DNA polymerases alpha and beta in repair of both photoproducts and AAF by inhibiting repair with several independent antibodies specific to either DNA polymerases alpha or beta and then restoring repair by adding the appropriate purified polymerase. Repair is inhibited by aphidicolin at concentrations specific for blocking DNA polymerase alpha and dideoxynucleotide triphosphates at concentrations specific for inhibiting DNA polymerase beta.

Xenopus oocytes and oocyte nuclear extracts repair ultraviolet photoproducts on double-stranded (ds) DNA and replicate single-stranded (ss) to ds DNA. M13 ss DNA molecules containing cyclobutane pyrimidine dimers were maintained but not replicated in Xenopus oocytes yet were replicated in progesterone-matured oocytes. The replication arrest functioned only in cis. The replication arrest was alleviated by injection into oocytes of messenger RNAs encoding the prokaryotic mutagenesis proteins UmuD'C or MucA'B. These results may help explain how cells stabilize repair or replication events on DNA with unrepairable lesions.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.

Angiogenin is a 14.4-kDa human plasma protein with 65% homology to RNase A that retains the key active site residues and three of the four RNase A disulfide bonds. We demonstrate that recombinant angiogenin functions as a cytotoxic tRNA-specific RNase in cell-free lysates and when injected into Xenopus oocytes. Inhibition of protein synthesis by angiogenin correlates with degradation of endogenous oocyte tRNA. Exogenous, radiolabeled tRNA is also hydrolyzed by angiogenin, whereas oocyte rRNA and mRNA are not detectably degraded by angiogenin. Protein synthesis was restored to angiogenin-injected oocytes by injecting the RNase inhibitor RNasin plus total Xenopus or calf liver tRNAs, thereby demonstrating that the tRNA degradation induced by angiogenin was the sole cause of cytotoxicity. A similar tRNA-reversible inhibition of protein synthesis was seen in rabbit reticulocyte lysates. Angiogenin therefore appears to be a specific cellular tRNase, whereas five homologues in the RNase A superfamily lack angiogenin's specificity for tRNA. One of these homologues purified from human eosinophils, eosinophil-derived neurotoxin, nonspecifically degrades oocyte RNA similar to RNase A and is also cytotoxic at very low concentrations.