Search for Novel Remedies to Augment Radiation Resistance of Inhabitants of Fukushima and Chernobyl Disasters: Identifying DNA Repair Protein XRCC4 Inhibitors

Abstract

Two nuclear plant disasters occurring within a span of 25 years threaten health and genome integrity both in Fukushima and Chernobyl. Search for remedies capable of enhancing DNA repair efficiency and radiation resistance in humans appears to be a urgent problem for now. XRCC4 is an important enhancer in promoting repair pathway triggered by DNA double-strand break (DSB). In the context of radiation therapy, active XRCC4 could reduce DSB-mediated apoptotic effect on cancer cells. Hence, developing XRCC4 inhibitors could possibly enhance radiotherapy outcomes. In this study, we screened traditional Chinese medicine (TCM) database, TCM Database@Taiwan, and have identified three potent inhibitor agents against XRCC4. Through molecular dynamics simulation, we have determined that the protein-ligand interactions were focused at Lys188 on chain A and Lys187 on chain B. Intriguingly, the hydrogen bonds for all three ligands fluctuated frequently but were held at close approximation. The pi-cation interactions and ionic interactions mediated by o-hydroxyphenyl and carboxyl functional groups respectively have been demonstrated to play critical roles in stabilizing binding conformations. Based on these results, we reported the identification of potential radiotherapy enhancers from TCM. We further characterized the key binding elements for inhibiting the XRCC4 activities.     

Screening from the world's largest TCM database for inhibiting DNA repair protein XRCC4

Human XRCC4 protein is a key component of DNA double-strand break (DSB) repair pathway related to the diseases of stroke and cancer. Cancer cells being treated with the drugs that interfere with DSBs repair mechanism have shown increased radiosensitivity to ionising radiation. Therefore, the development of novel radiosensitiser for radiation therapy becomes important for cancer treatment. We screened from the world's largest traditional Chinese medicine (TCM) database and found potential TCM molecules that can dock at XRCC4 functional site. Among the selected potential TCM compounds, we specifically investigated the top-ranked molecules: glycyrrhizic acid and macedonoside C. The molecular docking and molecular dynamics simulations on these compounds show similar location with high predicted binding affinity. Both compounds form continuous interaction with Lys188 and Arg192 of chain C and Lys187 and Lys190 of chain D. All these protein residues are required to form key hydrophobic interactions with other components participating in DNA repair. We suggested both glycyrrhizic acid and macedonoside C as potential lead compounds for inhibiting XRCC4.     

XRCC4 protein interactions with XRCC4-like factor (XLF) create an extended grooved scaffold for DNA ligation and double strand break repair

The XRCC4-like factor (XLF)-XRCC4 complex is essential for nonhomologous end joining, the major repair pathway for DNA double strand breaks in human cells. Yet, how XLF binds XRCC4 and impacts nonhomologous end joining functions has been enigmatic. Here, we report the XLF-XRCC4 complex crystal structure in combination with biophysical and mutational analyses to define the XLF-XRCC4 interactions. Crystal and solution structures plus mutations characterize alternating XRCC4 and XLF head domain interfaces forming parallel super-helical filaments. XLF Leu-115 ("Leu-lock") inserts into a hydrophobic pocket formed by XRCC4 Met-59, Met-61, Lys-65, Lys-99, Phe-106, and Leu-108 in synergy with pseudo-symmetric β-zipper hydrogen bonds to drive specificity. XLF C terminus and DNA enhance parallel filament formation. Super-helical XLF-XRCC4 filaments form a positively charged channel to bind DNA and align ends for efficient ligation. Collective results reveal how human XLF and XRCC4 interact to bind DNA, suggest consequences of patient mutations, and support a unified molecular mechanism for XLF-XRCC4 stimulation of DNA ligation.     

Tetramerization and DNA ligase IV interaction of the DNA double-strand break repair protein XRCC4 are mutually exclusive

The XRCC4 protein is of critical importance for the repair of broken chromosomal DNA by non-homologous end joining (NHEJ). The absence of XRCC4 abolishes chromosomal NHEJ almost completely. One reason for this severe phenotype is that XRCC4 binds and modulates the stability and activity of the NHEJ-specific ligase, DNA ligase IV. XRCC4 in solution is in equilibrium between the dimeric and tetrameric forms. Previous structural studies have shown that the interface between dimers is located in the same region as that implicated in DNA ligase IV interaction. With the use of equilibrium sedimentation analysis, we show here that only the XRCC4 dimer can associate with DNA ligase IV, forming a monodisperse complex of 2:1 stoichiometry in solution. In addition, physical analysis of XRCC4/DNA ligase IV complex formation, combined with mutational analysis of XRCC4, indicates that tetramerization and DNA ligase IV binding are mutually exclusive. We propose that the putative function of the XRCC4 tetramer is distinct from its DNA ligase IV-associated function.     

XRCC1 and DNA polymerase beta interaction contributes to cellular alkylating-agent resistance and single-strand break repair

X-ray cross complementing 1 (XRCC1) protein has been suggested to bind to DNA single-strand breaks (SSBs) and organize protein interactions that facilitate efficient DNA repair. Using four site-specifically modified human XRCC1 mutant expression systems and functional complementation assays in Chinese hamster ovary (CHO) XRCC1-deficient EM9 cells, we evaluated the cellular contributions of XRCC1s proposed N-terminal domain (NTD) DNA binding and DNA polymerase beta (POLbeta) interaction activities. Results within demonstrate that the interaction with POLbeta is biologically important for alkylating agent resistance and SSB repair, whereas the proposed DNA binding function is not critical to these phenotypes. Our data favor a model where the interaction of XRCC1 with POLbeta contributes to efficient DNA repair in vivo, whereas its interactions with target DNA is biologically less relevant.     

DNA repair: relationship to drug and radiation resistance, metastasis and growth factors

DNA repair mechanisms are important for the recovery of both normal and malignant tissues from radiation and chemotherapy. Drug 'resistance' may merely reflect the similarity of cancer to normal tissues. Investigating the normal repair mechanisms by cloning human DNA repair genes will permit a much better comparison. Therapeutic inhibition of DNA repair may be possible with poly-ADP-ribose polymerase inhibitors. A differential effect may be obtained since less-differentiated cells have a higher poly-ADP-ribose polymerase activity. Clinical application of repair inhibitors can be achieved by using antimetabolites such as high-dose hydroxyurea which produces levels of 1-3 mmol litre -1/24 hours. The whole cell and tissue response to DNA damage is more complex than removal of adducts and joining strand breaks. DNA damage can result in an increase in growth-factor receptors, the release of soluble mediators that affect undamaged cells and stimulation of plasminogen activator. These changes may enhance growth and recovery as well as bypass or repair the damage. The generation of heterogeneity in a tumour population may be mediated by DNA rearrangements. Genetic instability is much higher in metastatic clones and a comparison of DNA strand-break repair in a metastatic and a non-metastatic line showed more rapid repair in the former. Aberrant use of DNA repair stimulated by growth factors may mediate tumour progression and heterogeneity as well as drug resistance.     

A potential copper-regulatory role for cytosolic expression of the DNA repair protein XRCC5

Copper (Cu) has a critical role in the generation of oxidative stress during neurodegeneration and cancer. Reactive oxygen species generated through abnormal elevation or deficiency of Cu can lead to lipid, protein, and DNA damage. Oxidation of DNA can induce strand breaks and is associated with altered cell fate including transformation or death. DNA repair is mediated through the action of the multimeric DNA-PK repair complex. The components of this complex are the Ku autoantigens, XRCC5 and XRCC6 (Ku80 and Ku70, respectively). How this repair complex responds to perturbed Cu homeostasis and Cu-mediated oxidative stress has not been investigated. We previously reported that XRCC5 expression is altered in response to cellular Cu levels, with low Cu inhibiting XRCC5 expression and high Cu levels enhancing expression. In this study we further investigated the interaction between XRCC5 and Cu. We report that cytosolic XRCC5 is increased in response to Cu, but not zinc, iron, or nickel, and the level of cytosolic XRCC5 correlates with protection against oxidative damage to DNA. These observations were made in both HeLa cells and fibroblasts. Cytosolic XRCC5 interacted with the Cu chaperone and detoxification protein human Atox1 homologue (HAH), and down regulation of XRCC5 expression using siRNA led to enhanced HAH expression when cells were exposed to Cu. XRCC5 could also be purified from cytosolic extracts using a Cu-loaded column. These findings provide further evidence that cytosolic XRCC5 has a key role in protection against DNA oxidation from Cu, through either direct sequestration or signaling through other Cu-detoxification molecules. Our findings have important implications for the development of therapeutic treatments targeting Cu in neurodegeneration and/or cancer.     

Glucose deprivation increases nuclear DNA repair protein Ku and resistance to radiation induced oxidative stress in human cancer cells

Recent studies have indicated that nutrient deprivation particularly glucose may play a major role in tumor cell tolerance to a generally oxidative stress environment in solid tumors. Here, we studied the impact of glucose deprivation on the response of human colon (HT29) and prostate (DU145) cancer cells to γ radiation. A significant decrease in intracellular glucose level was observed in glucose deprived cells as measured by bioreductive assay. The survival of HT29 and DU145 were increased by 30 and 100% respectively when these cells were exposed to γ radiation in the absence of glucose compared to that in the presence of glucose. In glucose depleted medium, glutathione (GSH), a free radical scavenger, content remained the same, and showed no correlation with the radiation resistance induced by glucose deprivation. Glucose regulated protein78 (GRP78), a stress response survival protein, was not significantly increased in cells deprived of glucose for 4 h compared to those cells in glucose. DNA repair protein Ku, which is known to play a major role in cellular resistance to radiation, was significantly increased in glucose deprived cancer cells that showed enhanced radiation resistance. These results have demonstrated, for the first time, that glucose deprivation mediated stress increased the expression of nuclear Ku and resistance to radiation induced oxidative stress in human cancer cells. The additional resistance caused by glucose deprivation in cancer cells has clinical significance since solid tumors are known to have low level of glucose due to diffusion limited blood supply and higher metabolic activity. Copyright © 2009 John Wiley & Sons, Ltd.     

Saccharomyces cerevisiae LIF1: a function involved in DNA double-strand break repair related to mammalian XRCC4.

Saccharomyces cerevisiae DNA ligase IV (LIG4) has been shown previously to be involved in non-homologous DNA end joining and meiosis. The homologous mammalian DNA ligase IV interacts with XRCC4, a protein implicated in V(D)J recombination and double-strand break repair. Here, we report the discovery of LIF1, a S.cerevisiae protein that strongly interacts with the C-terminal BRCT domain of yeast LIG4. LIG4 and LIF1 apparently occur as a heterodimer in vivo. LIF1 shares limited sequence homology with mammalian XRCC4. Disruption of the LIF1 gene abolishes the capacity of cells to recircularize transformed linearized plasmids correctly by non-homologous DNA end joining. Loss of LIF1 is also associated with conditional hypersensitivity of cells to ionizing irradiation and with reduced sporulation efficiency. Thus, with respect to their phenotype, lif1 strains are similar to the previously described lig4 mutants. One function of LIF1 is the stabilization of the LIG4 enzyme. The finding of a XRCC4 homologue in S.cerevisiae now allows for mutational analyses of structure-function relationships in XRCC4-like proteins to define their role in DNA double-strand break repair.     

Loss of DNA ligase IV prevents recognition of DNA by double-strand break repair proteins XRCC4 and XLF

The repair of DNA double-strand breaks by nonhomologous end-joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Central to the molecular mechanism of NHEJ is DNA ligase IV/XRCC4/XLF complex, which rejoins the DNA. During adenovirus (Ad5) infection, ligase IV is targeted for degradation in a process that requires expression of the viral E1B 55k and E4 34k proteins while XRCC4 and XLF protein levels remain unchanged. We show that in Ad5-infected cells, loss of ligase IV is accompanied by loss of DNA binding by XRCC4. Expression of E1B 55k and E4 34k was sufficient to cause loss of ligase IV and loss of XRCC4 DNA binding. Using ligase IV mutant human cell lines, we determined that the absence of ligase IV, and not expression of viral proteins, coincided with inhibition of DNA binding by XRCC4. In ligase IV mutant human cell lines, DNA binding by XLF was also inhibited. Expression of both wild-type and adenylation-mutant ligase IV in ligase IV-deficient cells restored DNA binding by XRCC4. These data suggest that the intrinsic DNA-binding activities of XRCC4 and XLF may be subject to regulation and are down regulated in human cells that lack ligase IV.     

Long term effects of Chernobyl contamination on DNA repair function and plant resistance to different biotic and abiotic stress factors

Thirty years after the Chernobyl explosion we still lack information regarding the genetic effects of radionuclide contamination on the plant population. For example, are plants adapting to the low dose of chronic ionising irradiation and showing improved resistance to radiation damage? Are they coping with changing/increased pathogenicity of fungi and viruses in the Chernobyl exclusion (ChE) zone? Are plant populations rapidly accumulating mutational load and should we expect rapid micro-evolutionary changes in plants in the Chernobyl area? This review will try to summarise the current knowledge on these aspects of plant genetics and ecology and draw conclusions on the importance of further studies in the area around Chernobyl.     

Absence of DNA ligase IV protein in XR-1 cells: evidence for stabilization by XRCC4

XR-1 is a CHO mutant cell line defective in double strand break repair and V(D)J recombination. These defects are due to a deletion of the XRCC4 gene which encodes a 38-kDa nuclear phosphoprotein. Recent studies have shown that XRCC4 interacts with and enhances the activity of DNA ligase IV in vitro. In this study we investigate the effect of the absence of XRCC4 on the level of DNA ligase IV in XR-1 cells. Western blot analysis indicates that levels of DNA ligase IV protein are almost undetectable in these cells, however, introduction of the XRCC4 cDNA into XR-1 resulted in a return to wild type levels of the protein. Furthermore, analysis of DNA ligase IV mRNA showed equivalent levels in both XR-1 and XRCC4 transfected XR-1 indicating that the altered level of DNA ligase IV is not due to a change in the expression of the gene. These data strongly suggest that an important function of XRCC4 is to stabilize the DNA ligase IV protein.     

XRCC1 interactions with multiple DNA glycosylases: a model for its recruitment to base excision repair

Repair of chemically modified bases in DNA is accomplished through base excision repair (BER). This pathway is initiated by a specific DNA glycosylase that recognizes and excises the altered base to yield an abasic (AP) site. After cleavage of the AP site by APE1, repair proceeds through re-synthesis and ligation steps. In mammalian cells, the XRCC1 protein, essential for the maintenance of genomic stability, is involved in both base excision and single-strand break repair. XRCC1 participates in the first step of BER by interacting with the human DNA glycosylases hOGG1 and NEIL1. To analyze the possibility of a general mechanism involving the interaction of XRCC1 with DNA glycosylases we used XRCC1 to pull-down DNA glycosylases activities from human cell extracts. XRCC1 co-purifies with DNA glycosylase activities capable of excising hypoxanthine and dihydrothymine, in addition to 8-oxoguanine, but not uracil. Biochemical analyses with the purified proteins confirmed the interactions between XRCC1 and MPG, hNTH1 or hNEIL2. Furthermore, XRCC1 stimulates the activities of these enzymes. In vivo localization studies show that after genotoxic treatments these DNA glycosylases can be found associated with XRCC1 foci. Our results support a BER model in which XRCC1 is recruited to the repair of alkylated or oxidized bases by the enzyme recognizing the lesion. XRCC1 would then coordinate the subsequent enzymatic steps and modulate the activities of all the proteins involved.     

SUMO modification of human XRCC4 regulates its localization and function in DNA double-strand break repair

The nonhomologous end-joining (NHEJ) pathway is responsible for rejoining the majority of double-strand breaks in mammalian cells, including the programmed breaks introduced by V(D)J recombination. The regulation of the enzymatic activities associated with this recombination pathway is still largely unknown. Here we report that human XRCC4 (for X-ray cross-complementation group 4), a protein essential for NHEJ, is subject to posttranslational protein modification. The modifier peptide, SUMO, can be added to XRCC4 both in vitro and in vivo. The site of modification is mapped to lysine 210 by using specific mutagenesis. A protein mutated such that it cannot be SUMOylated remains localized in the cytoplasm rather than accumulating in the nucleus. Cells expressing only the mutated protein are radiation sensitive and fail to complete V(D)J recombination. Genetic fusion of the SUMO sequence to the C terminus of the mutant restores nuclear localization and radiation resistance. The modification may serve a regulatory role. Our finding fits with an emerging literature associating SUMO modification with the control of the repair and recombination associated with DNA breaks.     

Influence of polymorphisms in DNA repair genes XPD, XRCC1 and MGMT on DNA damage induced by gamma radiation and its repair in lymphocytes in vitro

DNA single-strand breaks (SSBs) were quantified by single-cell gel electrophoresis and micronucleated and apoptotic cells were quantified by microscopic assays in peripheral blood lymphocytes after irradiation on ice with 2 Gy of 60Co gamma radiation, and their association with polymorphisms of genes that encode proteins of different DNA repair pathways and influence cancer risk (XPD codon 312Asp --> Asn and 751Lys --> Gln, XRCC1 399Arg --> Gln, and MGMT 84Leu --> Phe) was studied. In unirradiated lymphocytes, SSBs were significantly more frequent in individuals older than the median age (52 years) (P = 0.015; n = 81), and the frequency of apoptotic or micronucleated cells was higher in individuals with alleles coding for Asn at XPD 312 or Gln at 751 (P = 0.030 or 0.023 ANOVA, respectively; n = 54). The only polymorphism associated with the background SSB level was MGMT 84Phe (P = 0.04, ANOVA; n = 66). After irradiation, SSB levels and repair parameters did not differ significantly with age or smoking habit. The SSB level varied more than twofold and the repair rate and level of unrepaired SSBs more than 10-fold between individuals. The presence of variant alleles coding for Asn at XPD 312 was associated with more radiation-induced SSBs (P = 0.014) and fewer unrepaired SSBs (P = 0.008), and the phenotype (> median induced SSBs/< median unrepaired SSBs) was seen in the majority of XPD 312Asn/Asn homozygotes; the odds ratio for variant homozygotes to show this phenotype was 5.2 (95% confidence interval 1.4-19.9). The hypothesis is discussed that XPD could participate in repair of ionizing radiation-induced DNA damage. While it cannot be excluded that the effects observed are due to cosegregating polymorphisms or that the responses of lymphocytes are not typical of other cell types, the results suggest that polymorphism of DNA repair genes, particularly XPD, is one factor implicated in the variability of responses to ionizing radiation between different individuals.     

Search for genes essential for pneumococcal transformation: the RADA DNA repair protein plays a role in genomic recombination of donor DNA

We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counterselection of mutants in 21 different genes during the challenge. Eight of the counterselected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants of seven of the eight remaining genes, i.e., spr0459-spr0460, spr0777, spr0838, spr1259-spr1260, and spr1357, resulted in reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation with chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and in DNA damage repair in S. pneumoniae.     

The Rad9 protein enhances survival and promotes DNA repair following exposure to ionizing radiation

Following DNA damage cells initiate cell cycle checkpoints to allow time to repair sustained lesions. Rad9, Rad1, and Hus1 proteins form a toroidal complex, termed the 9-1-1 complex, that is involved in checkpoint signaling. 9-1-1 shares high structural similarity to the DNA replication protein proliferating cell nuclear antigen (PCNA) and 9-1-1 has been shown in vitro to stimulate steps of the repair process known as long patch base excision repair. Using a system that allows conditional repression of the Rad9 protein in human cell culture, we show that Rad9, and by extension, the 9-1-1 complex, enhances cell survival, is required for efficient exit from G2-phase arrest, and stimulates the repair of damaged DNA following ionizing radiation. These data provide in vivo evidence that the human 9-1-1 complex participates in DNA repair in addition to its previously described role in DNA damage sensing.