Exposure to galactic cosmic radiation compromises DNA repair and increases the potential for oncogenic chromosomal rearrangement in bronchial epithelial cells.

Participants in deep space missions face protracted exposure to galactic cosmic radiation (GCR). In this setting, lung cancer is a significant component of the overall risk of radiation-exposure induced death. Here we investigate persistent effects of GCR exposure on DNA repair capacity in lung-derived epithelial cells, using an enzyme-stimulated chromosomal rearrangement as an endpoint. Replicate cell cultures were irradiated with energetic 48Ti ions (a GCR component) or reference γ-rays. After a six-day recovery, they were challenged by expression of a Cas9/sgRNA pair that creates double-strand breaks simultaneously in the EML4 and ALK loci, misjoining of which creates an EML4-ALK fusion oncogene. Misjoining was significantly elevated in 48Ti-irradiated populations, relative to the baseline rate in mock-irradiated controls. The effect was not seen in γ-ray irradiated populations exposed to equal or higher radiation doses. Sequence analysis of the EML4-ALK joints from 48Ti-irradiated cultures showed that they were far more likely to contain deletions, sometimes flanked by short microhomologies, than equivalent samples from mock-irradiated cultures, consistent with a shift toward error-prone alternative nonhomologous end joining repair. Results suggest a potential mechanism by which a persistent physiological effect of GCR exposure may increase lung cancer risk.

Galactic Cosmic Radiation Induces Persistent Epigenome Alterations Relevant to Human Lung Cancer.

Human deep space and planetary travel is limited by uncertainties regarding the health risks associated with exposure to galactic cosmic radiation (GCR), and in particular the high linear energy transfer (LET), heavy ion component. Here we assessed the impact of two high-LET ions 56Fe and 28Si, and low-LET X rays on genome-wide methylation patterns in human bronchial epithelial cells. We found that all three radiation types induced rapid and stable changes in DNA methylation but at distinct subsets of CpG sites affecting different chromatin compartments. The 56Fe ions induced mostly hypermethylation, and primarily affected sites in open chromatin regions including enhancers, promoters and the edges ("shores") of CpG islands. The 28Si ion-exposure had mixed effects, inducing both hyper and hypomethylation and affecting sites in more repressed heterochromatic environments, whereas X rays induced mostly hypomethylation, primarily at sites in gene bodies and intergenic regions. Significantly, the methylation status of 56Fe ion sensitive sites, but not those affected by X ray or 28Si ions, discriminated tumor from normal tissue for human lung adenocarcinomas and squamous cell carcinomas. Thus, high-LET radiation exposure leaves a lasting imprint on the epigenome, and affects sites relevant to human lung cancer. These methylation signatures may prove useful in monitoring the cumulative biological impact and associated cancer risks encountered by astronauts in deep space.

Understanding cancer development processes after HZE-particle exposure: roles of ROS, DNA damage repair and inflammation.

During space travel astronauts are exposed to a variety of radiations, including galactic cosmic rays composed of high-energy protons and high-energy charged (HZE) nuclei, and solar particle events containing low- to medium-energy protons. Risks from these exposures include carcinogenesis, central nervous system damage and degenerative tissue effects. Currently, career radiation limits are based on estimates of fatal cancer risks calculated using a model that incorporates human epidemiological data from exposed populations, estimates of relative biological effectiveness and dose-response data from relevant mammalian experimental models. A major goal of space radiation risk assessment is to link mechanistic data from biological studies at NASA Space Radiation Laboratory and other particle accelerators with risk models. Early phenotypes of HZE exposure, such as the induction of reactive oxygen species, DNA damage signaling and inflammation, are sensitive to HZE damage complexity. This review summarizes our current understanding of critical areas within the DNA damage and oxidative stress arena and provides insight into their mechanistic interdependence and their usefulness in accurately modeling cancer and other risks in astronauts exposed to space radiation. Our ultimate goals are to examine potential links and crosstalk between early response modules activated by charged particle exposure, to identify critical areas that require further research and to use these data to reduced uncertainties in modeling cancer risk for astronauts. A clearer understanding of the links between early mechanistic aspects of high-LET response and later surrogate cancer end points could reveal key nodes that can be therapeutically targeted to mitigate the health effects from charged particle exposures.

Gene amplification and associated loss of 5' regulatory sequences of CoAA in human cancers.

CoAA is an RRM-containing transcriptional coactivator that stimulates transcriptional activation and regulates alternative splicing. We show that the CoAA gene is amplified at the chromosome 11q13 locus in a subset of primary human cancers including non-small cell lung carcinoma, squamous cell skin carcinoma and lymphoma. Analysis of 42 primary tumors suggests that CoAA amplifies independently from the CCND1 locus. Detailed mapping of three CoAA amplicons reveals that the amplified CoAA gene is consistently located at the 5' boundaries of the amplicons. The CoAA coding and basal promoter sequences are retained within the amplicons but upstream silencing sequences are lost. CoAA protein is overexpressed in tumors containing the amplified CoAA gene. RNA dot blot analysis of 100 cases of primary tumors suggests elevated CoAA mRNA expression. CoAA positively regulates its own basal promoter in transfection assays. Thus, gene amplification, loss of silencing sequence and positive feedback regulation may lead to drastic upregulation of CoAA protein. CoAA has transforming activities when tested in soft agar assays, and CoAA is homologous to oncoproteins EWS and TLS, which regulate alternative splicing. These data imply that CoAA may share a similar oncogenic mechanism with oncogene EWS and that CoAA deregulation may alter the alternative splicing of target genes.

Autoantibodies against DNA double-strand break repair proteins.

Autoantibodies against cellular components are commonly present in sera from patients with systemic rheumatic diseases and may play an important role in pathogenesis. The Ku protein was recognized 20 years ago as a major target of autoantibodies in a subset of Japanese patients with scleroderma-polymyositis overlap syndrome, and anti-Ku antibodies have since been shown to occur in 10-20% of patients with these and other systemic rheumatic diseases, including systemic lupus erythematosus. Ku functions physiologically in the repair of DNA double-strand breaks, where it carries out the initial recognition of damaged DNA ends. The three dimensional structure of the Ku-DNA complex has recently been solved, and helps illuminate the relationship between the autoimmune epitopes and other features of the protein. In addition to Ku, three other polypeptides in the same DNA repair pathway have more recently been identified as autoantigens: the DNA-dependent protein kinase catalytic subunit, DNA ligase IV, and XRCC4. Two hypotheses have been invoked to explain the ability of these proteins to elicit an autoimmune response in susceptible individuals. One is that DNA damage induces formation of nucleoprotein complexes that present novel composite or conformational epitopes. The other is that cleavage of these proteins by caspases or Granzyme B leads to presentation of immunocryptic peptides capable of stimulating autoreactive T lymphocytes. In the case of DNA double-strand break repair proteins, there is evidence that both of these mechanisms may be at work. Because of their role in the maintenance of genome stability, DNA double-strand break repair proteins have been the subject of intense study, and a wealth of new structural, biochemical and functional information makes them excellent models for investigation of the humoral autoimmune response.

A method to detect particle-specific antibodies against Ku and the DNA-dependent protein kinase catalytic subunit in autoimmune sera.

Sera from patients with systemic lupus erythematosus, polymyositis, scleroderma, and mixed connective tissue disease are frequently characterized by the presence of high levels of autoantibodies directed against linked sets of nuclear proteins. One of these autoantigen systems is made up of Ku and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), proteins that are essential for double-strand DNA break repair and for the related process of V(D)J recombination. Ku and DNA-PKcs bind avidly to DNA ends in vivo and in vitro and form an active protein kinase complex. One hypothesis is that this assembled nucleoprotein particle, rather than its component proteins, is a primary trigger for the autoimmune response and thus a major target for the resulting autoantibodies. To screen for particle-specific antibodies, we developed an assay in which the fully native nucleoprotein particle is reconstituted in vitro and is tethered to the surface of an ELISA plate via a streptavidin-biotin linkage. These particles are recognized efficiently by monoclonal antibodies and by autoantibodies present in patient sera. The assay may detect a broader spectrum of epitopes than a conventional ELISA in which Ku and DNA-PKcs are adsorbed directly to a plastic surface. The method will be advantageous for high-throughput screening for antibodies and other ligands that bind the assembled DNA-dependent protein kinase complex.

Distinct roles for Ku protein in transcriptional reinitiation and DNA repair.

Transcriptional reinitiation is a distinct phase of the RNA polymerase II transcription cycle. Prior work has shown that reinitiation is deficient in nuclear extracts from Chinese hamster ovary cells lacking the 80-kDa subunit of Ku, a double-strand break repair protein, and that activity is rescued by expression of the corresponding cDNA. We now show that Ku increases the amount or availability of a soluble factor that is limiting for reinitiation, that the factor increases the number of elongation complexes associated with the template at all times during the reaction, and that the factor itself does not form a tight complex with DNA. The factor may consist of a preformed complex of transcription proteins that is stabilized by Ku. A Ku mutant, lacking residues 687-728 in the 80-kDa subunit, preferentially suppresses transcription in Ku-containing extracts, suggesting that Ku interacts directly with proteins required for reinitiation. The Ku mutant functions normally in a DNA end-joining system, indicating that the functions of Ku in transcription and repair are genetically separable. Based on our results, we present a model in which Ku is capable of undergoing a switch between a transcription factor-associated and a repair-active state.

DNA ligase IV and XRCC4 form a stable mixed tetramer that functions synergistically with other repair factors in a cell-free end-joining system.

Repair of DNA double-strand breaks in mammalian cells occurs via a direct nonhomologous end-joining pathway. Although this pathway can be studied in vivo and in crude cell-free systems, a deeper understanding of the mechanism requires reconstitution with purified enzymes. We have expressed and purified a complex of two proteins that are critical for double-strand break repair, DNA ligase IV (DNL IV) and XRCC4. The complex is homogeneous, with a molecular mass of about 300,000 Da, suggestive of a mixed tetramer containing two copies of each polypeptide. The presence of multiple copies of DNL IV was confirmed in an experiment where different epitope-tagged forms of DNL IV were recovered simultaneously in the same complex. Cross-linking suggests that an XRCC4.XRCC4 dimer interface forms the core of the tetramer, and that the DNL IV polypeptides are in contact with XRCC4 but not with one another. Purified DNL IV.XRCC4 complex functioned synergistically with Ku protein, the DNA-dependent protein kinase catalytic subunit, and other repair factors in a cell-free end-joining assay. We suggest that a dyad-symmetric DNL IV.XRCC4 tetramer bridges the two ends of the broken DNA and catalyzes the coordinate ligation of the two DNA strands.

Identification of a human T-cell leukemia virus type I tax peptide in contact with DNA.

The human T-cell leukemia virus Tax protein directs binding of a host factor, cAMP response element binding protein, to an extended recognition sequence in the proviral promoter. Prior cross-linking experiments have revealed that Tax makes restricted contact with this DNA at two symmetric positions, 14 nucleotides apart on opposite strands of the DNA. Tax lacks a conventional DNA binding domain, and the sequences in Tax that are in contact with DNA have not been previously identified. Analysis of cross-linked peptides now shows that the contact occurs between Tax residues 89 and 110, corresponding to a protease-sensitive linker joining two protein structural domains. The linker assumes a protease-resistant conformation in the cross-linked complex. Point mutations within the linker prevent cross-linking and interfere with Tax function. These data suggest that entry of Tax into the ternary complex may be coupled to folding of an unstructured protein domain, which then makes base-specific contacts with DNA.

Geometry of a complex formed by double strand break repair proteins at a single DNA end: recruitment of DNA-PKcs induces inward translocation of Ku protein.

Ku protein and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are essential components of the double-strand break repair machinery in higher eukaryotic cells. Ku protein binds to broken DNA ends and recruits DNA-PKcs to form an enzymatically active complex. To characterize the arrangement of proteins in this complex, we developed a set of photocross-linking probes, each with a single free end. We have previously used this approach to characterize the contacts in an initial Ku-DNA complex, and we have now applied the same technology to define the events that occur when Ku recruits DNA-PKcs. The new probes allow the binding of one molecule of Ku protein and one molecule of DNA-PKcs in a defined position and orientation. Photocross-linking reveals that DNA-PKcs makes direct contact with the DNA termini, occupying an approximately 10 bp region proximal to the free end. Characterization of the Ku protein cross-linking pattern in the presence and absence of DNA-PKcs suggests that Ku binds to form an initial complex at the DNA ends, and that recruitment of DNA-PKcs induces an inward translocation of this Ku molecule by about one helical turn. The presence of ATP had no effect on protein-DNA contacts, suggesting that neither DNA-PK-mediated phosphorylation nor a putative Ku helicase activity plays a role in modulating protein conformation under the conditions tested.

Human RNA helicase A is a lupus autoantigen that is cleaved during apoptosis.

Proteolytic cleavage by caspases is the central event in cells undergoing apoptosis. Cleaved proteins are often targeted by autoantibodies, suggesting that the cleavage of self Ags enhances immunogenicity and is prone to induce an autoimmune response. We found autoantibodies that immunoprecipitated a 140-kDa RNA-associated protein, provisionally designated Pa, in 11 of 350 patient sera that were positive for antinuclear Abs in an immunofluorescence test. The Pa protein gave rise to three fragments with m.w. ranging from 120-130 kDa during anti-Fas-activated apoptosis. Pure caspase-3 cleaved the Pa protein into a 130-kDa fragment corresponding to the largest of these three products. Peptide sequence analysis of a tryptic digest from immunoaffinity-purified Pa showed 100% identity to human RNA helicase A (RHA). The identity of Pa with RHA was further confirmed by immunoblotting with rabbit anti-RHA Ab using anti-Pa immunoprecipitates as substrates. All 10 anti-RHA-positive patients who were clinically analyzed were diagnosed as having systemic lupus erythematosus, and 7 of them had lupus nephritis. RHA is a multifunctional protein with roles in cellular RNA synthesis and processing. Inactivation of RHA by cleavage may be an important part of the process leading to programmed cell death. The cleaved RHA fragments that are produced during apoptosis may trigger an autoimmune response in systemic lupus erythematosus.

Photocross-linking of an oriented DNA repair complex. Ku bound at a single DNA end.

Ku protein binds broken DNA ends, triggering a double-strand DNA break repair pathway. The spatial arrangement of the two Ku subunits in the initial Ku-DNA complex, when the Ku protein first approaches the broken DNA end, is not well defined. We have investigated the geometry of the complex using a novel set of photocross-linking probes that force Ku protein to be constrained in position and orientation, relative to a single free DNA end. Results suggest that this complex is roughly symmetric and that both Ku subunits make contact with an approximately equal area of the DNA. The complex has a strongly preferred orientation, with Ku70-DNA backbone contacts located proximal and Ku80-DNA backbone contacts located distal to the free end. Ku70 also contacts functional groups in the major groove proximal to the free end. Ku80 apparently does not make major groove contacts. Results are consistent with a model where the Ku70 and Ku80 subunits contact the major and minor grooves of DNA, respectively.

DNA-dependent protein kinase protects against heat-induced apoptosis.

Purified heat shock transcription factor 1 (HSF1) binds to both the regulatory and catalytic components of the DNA-dependent protein kinase (DNA-PK). This observation suggests that DNA-PK may have a physiological role in the heat shock response. To investigate this possibility, we performed a comparison of cell lines that were deficient in either the Ku protein or the DNA-PK catalytic subunit versus the same cell lines that had been rescued by the introduction of a functional gene. DNA-PK-negative cell lines were up to 10-fold more sensitive to heat-induced apoptosis than matched DNA-PK-positive cell lines. There may be a regulatory interaction between DNA-PK and HSF1 in vivo, because constitutive overexpression of HSF1 sensitized the DNA-PK-positive cells to heat but had no effect in DNA-PK-negative cells. The initial burst of hsp70 mRNA expression was similar in DNA-PK-negative and -positive cell lines, but the DNA-PK-negative cells showed an attenuated rate of mRNA synthesis at later times and, in some cases, lower heat shock protein expression. These findings provide evidence for an antiapoptotic function of DNA-PK that is experimentally separable from its mechanical role in DNA double strand break repair.

Nuclear extracts lacking DNA-dependent protein kinase are deficient in multiple round transcription.

We have compared levels of in vitro transcription in nuclear extracts from DNA-dependent protein kinase (DNA-PK)-deficient and DNA-PK-containing Chinese hamster ovary cell lines. DNA-PK-deficient cell lines are radiosensitive mutants lacking either the catalytic subunit or the 80-kDa subunit of the Ku protein regulatory component. Extracts from DNA-PK-deficient cell lines had a 2-7-fold decrease in the level of in vitro transcription when compared with matched controls. This decrease was observed with several promoters. Transcription could be restored to either of the deficient extracts by addition of small amounts of extract from the DNA-PK-containing cell lines. Transcription was not restored by addition of purified DNA-PK catalytic subunit, Ku protein, or individually purified general transcription factors. We conclude that extracts from DNA-PK-deficient cells lack a positively acting regulatory factor or a complex of factors not readily reconstituted with individual proteins. We have also investigated the mechanistic defect in the deficient extracts and have found that the observed differences in transcription levels between Ku-positive and Ku-negative cell lines can be attributed solely to a greater ability of the Ku-positive nuclear extracts to carry out secondary initiation events subsequent to the first round of transcription.

Human autoantibodies recognizing a native macromolecular structure composed of Sm core proteins in U small nuclear RNP particles.

OBJECTIVE: Monoclonal antibody (mAb) F78 recognizes a heat-labile particle composed of Sm core proteins designated F78P. The objective of this study was to identify human autoantibodies recognizing the conformational structure of F78P. METHODS: Immunoblots using HeLa cell extracts without heating prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to identify autoantibodies recognizing F78P. To confirm reactivities with F78P, immunoprecipitates of mAb F78 were used as a substrate for immunoblots. To identify reactivities against the F78P structure in classic anti-Sm-positive sera, autoantibodies to individual Sm core proteins were absorbed with purified U1 small nuclear RNP before immunoblotting. RESULTS: We identified 2 sera that, like F78, recognized only F78P and not its component polypeptides. When classic anti-Sm antibodies were preabsorbed, the presence of F78-like, particle-specific antibodies was revealed in all of the anti-Sm-positive sera tested. CONCLUSION: Autoantibodies against the F78P structure were commonly present in sera from patients with systemic rheumatic diseases, often in combination with4=1998 M autoantibodies.

Specific regions of contact between human T-cell leukemia virus type I Tax protein and DNA identified by photocross-linking.

The human T-cell leukemia virus type I Tax protein forms a ternary complex on DNA in association with a host factor, the cyclic AMP response element-binding protein (CREB). An understanding of the precise geometry of this complex has been elusive. We have used photocross-linking to investigate Tax-DNA contacts. Our data show that Tax contacts the DNA at two symmetric positions 14 nucleotides apart on either side of the Tax responsive element. The presence of symmetric, widely separated regions of contact suggests that at least two molecules of Tax are present in the complex. Mapping the contacts onto a three-dimensional model of the CREB-DNA binary complex shows that they lie on the same face of the DNA near the regions where the N termini of the CREB bZIP domains enter the major groove. This location correlates well with previous evidence that CREB amino acid residues immediately N-terminal to the bZIP domain are crucial for the formation of the ternary complex. The limited number of cross-links observed suggests that contacts are primarily with the phosphate backbone and does not support the idea that a major structural element of the Tax protein inserts into the major or minor grooves of the DNA.

Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids.

The Ku protein-DNA-dependent protein kinase system is one of the major pathways by which cells of higher eukaryotes respond to double-strand DNA breaks. The components of the system are evolutionarily conserved and homologs are known from a number of organisms. The Ku protein component binds directly to DNA ends and may help align them for ligation. Binding of Ku protein to DNA also nucleates formation of an active enzyme complex containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The interaction between Ku protein, DNA-PKcs and nucleic acids has been extensively investigated. This review summarizes the results of these biochemical investigations and relates them to recent molecular genetic studies that reveal highly characteristic repair and recombination defects in mutant cells lacking Ku protein or DNA-PKcs.

Characterization of the RNA binding properties of Ku protein.

Ku protein, a heterodimer of 70 and 83 kDa polypeptides, is the regulatory component of the DNA-dependent protein kinase (DNA-PK). Ku protein binds to DNA ends and is essential for DNA double-strand break repair and V(D)J recombination. Although there is some evidence that Ku protein also binds RNA, its RNA binding properties have not been systematically explored. In the present study, Ku-binding RNAs were identified using systematic evolution of ligands by exponential enrichment (SELEX) technology. These RNAs were assigned to three classes based on common sequence motifs. Most of the selected RNAs bound to Ku protein with a Kd < or = 2 nM, comparable to the affinity of DNA fragments for Ku protein under similar conditions. Many of the RNAs inhibited DNA-PK activity by competing with DNA for a common binding site in Ku protein. None of several RNAs that were tested activated DNA-PK in the absence of DNA. The identification of diverse RNAs that bind avidly to Ku protein is consistent with the idea that natural RNAs may serve as modulators of DNA-PK activity. Moreover, the RNAs identified in this study may have utility as tools for experimental manipulation of DNA double-strand break repair activity in cells and cell extracts.

Heat shock transcription factor 1 binds selectively in vitro to Ku protein and the catalytic subunit of the DNA-dependent protein kinase.

Heat shock transcription factor 1 (HSF1) functions as the master regulator of the heat shock response in eukaryotes. We have previously shown that, in addition to its role as a transcription factor, HSF1 stimulates the activity of the DNA-dependent protein kinase (DNA-PK). DNA-PK is composed of two components: a 460-kDa catalytic subunit and a 70- and 86-kDa heterodimeric regulatory component, also known as the Ku protein. We report here that HSF1 binds specifically to each of the two components of DNA-PK. Binding occurs in the absence of DNA. The complex with the Ku protein is stable and forms at a stoichiometry close to unity between the Ku protein heterodimer and the active HSF1 trimer. The binding is blocked by antibodies against HSF1. Our results show that HSF1 also binds directly, but more weakly, to the catalytic subunit of DNA-PK. Both interactions are dependent on a specific region within the HSF1 regulatory domain. This sequence is necessary but not sufficient for HSF1 stimulation of DNA-PK activity. The ability of HSF1 to interact with both components of DNA-PK provides a potential mechanism for the activation of DNA-PK in response to heat and other forms of stress.

Analysis of the phosphorylation of human heat shock transcription factor-1 by MAP kinase family members.

The activation of heat shock transcription factor-1 (HSF-1) after treatment of mammalian cells with stresses such as heat shock, heavy metals, or ethanol induces the synthesis of heat shock proteins. HSF-1 is phosphorylated at normal growth temperature and is hyperphosphorylated upon stress. We recently presented evidence that HSF-1 can be phosphorylated by the mitogen activated protein kinase, ERK1, and that such phosphorylation appears to negatively regulate the activity of HSF-1. In this report, we have tested the ability of ERK1 to phosphorylate various HSF-1 deletion mutants. Our results show that ERK1 phosphorylation is dependent on a region of HSF-1 extending from amino acids 280 to 308. This region contains three serine residues that are potential ERK1 phosphorylation sites. The region falls within a previously defined regulatory domain of HSF-1. The possibility of protein kinases other than ERK1 phosphorylating HSF-1 was also examined using in-gel kinase assays. The results show that HSF-1 can be phosphorylated in a ras-dependent manner by other members of the MAP kinase family such as JNK and p38 protein kinases and possibly others.