Genetic factors in individual radiation sensitivity

Cancer risk and radiation sensitivity are often associated with alterations in DNA repair, cell cycle, or apoptotic pathways. Interindividual variability in mutagen or radiation sensitivity and in cancer susceptibility may also be traced back to polymorphisms of genes affecting e.g. DNA repair capacity. We studied possible associations between 70 polymorphisms of 12 DNA repair genes with basal and initial DNA damage and with repair thereof. We investigated DNA damage induced by ionizing radiation in lymphocytes isolated from 177 young lung cancer patients and 169 cancer-free controls. We also sought replication of our findings in an independent sample of 175 families (in total 798 individuals). DNA damage was assessed by the Olive tail moment (OTM) of the comet assay. DNA repair capacity (DRC) was determined for 10, 30 and, 60 min of repair.Genes involved in the single-strand-repair pathway (SSR; like XRCC1 and MSH2) as well as genes involved in the double-strand-repair pathway (DSR; like RAD50, XRCC4, MRE11 and ATM) were found to be associated with DNA damage. The most significant association was observed for marker rs3213334 (p = 0.005) of XRCC1 with basal DNA damage (B), in both cases and controls. A clear additive effect on the logarithm of OTM was identified for the marker rs1001581 of the same LD-block (p = 0.039): B_CC = −1.06 (95%-CI: −1.16 to −0.96), B_CT = −1.02 (95%-CI: −1.11 to −0.93) and B_TT = −0.85 (95%-CI: −1.01 to −0.68). In both cases and controls, we observed significantly higher DNA basal damage (p = 0.007) for carriers of the genotype AA of marker rs2237060 of RAD50 (involved in DSR). However, this could not be replicated in the sample of families (p = 0.781). An alteration to DRC after 30 min of repair with respect to cases was observed as borderline significant for marker rs611646 of ATM (involved in DSR; p = 0.055), but was the most significant finding in the sample of families (p = 0.009).Our data indicate that gene variation impacts measurably on DNA damage and repair, suggesting at least a partial contribution to radiation sensitivity and lung cancer susceptibility.     

Interaction between APC and Fen1 during breast carcinogenesis

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER – adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) – promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.     

BRCA1: Beyond double-strand break repair

Since its discovery, the BRCA1 tumor suppressor has been shown to play a role in multiple DNA damage response pathways. Here, we will review the involvement of BRCA1 in base-excision DNA repair and highlight its clinical implications.     

ARTD1 (PARP1) activation and NAD+ in DNA repair and cell death

Nicotinamide adenine dinucleotide, NAD⁺, is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD⁺ and NADH). NAD⁺ is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalyzed by the sirtuins (SIRTs) which use NAD⁺ as a substrate. In this review, we highlight the significance of NAD⁺ catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by ARTD1 activation and energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked, are dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischaemia/reperfusion.     

Structural features,evolutionary relationships,and transcriptional regulation of C-type lectin-domain proteins in Manduca sexta

C-type lectins (CTLs) are a large family of Ca²⁺-dependent carbohydrate-binding proteins recognizing various glycoconjugates and functioning primarily in immunity and cell adhesion. We have identified 34 CTLDP (for CTL-domain protein) genes in the Manduca sexta genome, which encode proteins with one to three CTL domains. CTL-S1 through S9 (S for simple) have one or three CTL domains; immulectin-1 through 19 have two CTL domains; CTL-X1 through X6 (X for complex) have one or two CTL domains along with other structural modules. Nine simple CTLs and seventeen immulectins have a signal peptide and are likely extracellular. Five complex CTLs have both an N-terminal signal peptide and a C-terminal transmembrane region, indicating that they are membrane anchored. Immulectins exist broadly in Lepidoptera and lineage-specific gene duplications have generated three clusters of fourteen genes in the M. sexta genome, thirteen of which have similar expression patterns. In contrast to the family expansion, CTL-S1∼S6, S8, and X1∼X6 have 1:1 orthologs in at least four lepidopteran/dipteran/coleopteran species, suggestive of conserved functions in a wide range of holometabolous insects. Structural modeling suggests the key residues for Ca²⁺-dependent or independent binding of certain carbohydrates by CTL domains. Promoter analysis identified putative κB motifs in eighteen of the CTL genes, which did not have a strong correlation with immune inducibility in the mRNA or protein levels. Together, the gene identification, sequence comparisons, structure modeling, phylogenetic analysis, and expression profiling establish a solid foundation for future studies of M. sexta CTL-domain proteins.     

DR-GAS: A database of functional genetic variants and their phosphorylation states in human DNA repair systems

We present DR-GAS¹, a unique, consolidated and comprehensive DNA repair genetic association studies database of human DNA repair system. It presents information on repair genes, assorted mechanisms of DNA repair, linkage disequilibrium, haplotype blocks, nsSNPs, phosphorylation sites, associated diseases, and pathways involved in repair systems. DNA repair is an intricate process which plays an essential role in maintaining the integrity of the genome by eradicating the damaging effect of internal and external changes in the genome. Hence, it is crucial to extensively understand the intact process of DNA repair, genes involved, non-synonymous SNPs which perhaps affect the function, phosphorylated residues and other related genetic parameters. All the corresponding entries for DNA repair genes, such as proteins, OMIM IDs, literature references and pathways are cross-referenced to their respective primary databases. DNA repair genes and their associated parameters are either represented in tabular or in graphical form through images elucidated by computational and statistical analyses. It is believed that the database will assist molecular biologists, biotechnologists, therapeutic developers and other scientific community to encounter biologically meaningful information, and meticulous contribution of genetic level information towards treacherous diseases in human DNA repair systems. DR-GAS is freely available for academic and research purposes at: http://www.bioinfoindia.org/drgas.     

EWGWS insert in Plasmodium falciparum ookinete surface enolase is involved in binding of PWWP containing peptides: Implications to mosquito midgut invasion by the parasite

There are multiple stages in the life cycle of Plasmodium that invade host cells. Molecular machinery involved is such host–pathogen interactions constitute excellent drug targets and/or vaccine candidates. A screen using a phage display library has previously demonstrated presence of enolase on the surface of the Plasmodium ookinete. Phage-displayed peptides that bound to the ookinete contained a conserved motif (PWWP) in their sequence. Here, direct binding of these peptides with recombinant Plasmodium falciparum enolase (rPfeno) was investigated. These peptides showed specific binding to rPfeno, but failed to bind to other enolases. Plasmodium spp enolases are distinct in having an insert of five amino acids (¹⁰⁴EWGWS¹⁰⁸) that is not found in host enolases. The possibility of this insert being the recognition motif for the PWWP containing peptides was examined, (i) by comparing the binding of the peptides with rPfeno and a deletion variant Δ-rPfeno lacking ¹⁰⁴EWGWS¹⁰⁸, (ii) by measuring the changes in proton chemical shifts of PWWP peptides on binding to different enolases and (iii) by inter-molecular docking experiment to locate the peptide binding site. Results from these studies showed that the pentapeptide insert of Pfeno indeed constitutes the binding site for the PWWP domain containing peptide ligands. Search for sequences homologous to phage displayed peptides among peritrophic matrix proteins resulted in identification of perlecan, laminin, peritrophin and spacran. The possibility of these PWWP domain-containing proteins in the peritrophic matrix of insect gut to interact with ookinete cell surface enolase and facilitate the invasion of mosquito midgut epithelium is discussed.     

Cytotoxic mechanism of novel compound jiangxienone from Cordyceps jiangxiensis against cancer cells involving DNA damage response pathway

Jiangxienone is a novel compound recently purified from the traditional Chinese medicinal mushroom Cordyceps jiangxiensis and was reported to show potent cytotoxicity against cancer cells. However, its mechanism of action remains unclear. In this work, the underlying mechanism of jiangxienone against human gastric cancer cells HGC-27 was investigated using whole-genome microarray. The results demonstrated that jiangxienone significantly decreased cell population of various human cancer cell lines, while slightly inhibited the colony formation of stromal cells from murine marrow even at a high concentration. Differential gene expression profiling indicated that the cytotoxic action of jiangxienone against HGC-27 is closely related to the DNA damage response pathway, which was evident by the identification of 23 DNA damage response-associated genes, such as XRCC4/5/6, NBS1, RAD51, and BRCA1/2. By using gel retardation assays, UV absorption spectrometry and single-cell gel electrophoresis, it was found that jiangxienone could bind to DNA and inhibit cancer cell growth. The above results indicated that the cytotoxic mechanism of jiangxienone against cancer cells was involved in the DNA damage response pathway. The findings will be helpful to the development of useful cancer chemopreventive compounds from C. jiangxiensis.     

The complexity of DNA double strand break is a crucial factor for activating ATR signaling pathway for G2/M checkpoint regulation regardless of ATM function

DNA double strand break (DSB) repair pathway choice following ionizing radiation (IR) is currently an appealing research topic, which is still largely unclear. Our recent paper indicated that the complexity of DSBs is a critical factor that enhances DNA end resection. It has been well accepted that the RPA-coated single strand DNA produced by resection is a signaling structure for ATR activation. Therefore, taking advantage of high linear energy transfer (LET) radiation to effectively produce complex DSBs, we investigated how the complexity of DSB influences the function of ATR pathway on the G2/M checkpoint regulation. Human skin fibroblast cells with or without ATM were irradiated with X rays or heavy ion particles, and dual-parameter flow cytometry was used to quantitatively assess the mitotic entry at early period post radiation by detecting the cells positive for phosphor histone H3. In ATM-deficient cells, ATR pathway played a pivotal role and functioned in a dose- and LET-dependent way to regulate the early G2/M arrest even as low as 0.2 Gy for heavy ion radiation, which indicated that ATR pathway could be rapidly activated and functioned in an ATM-independent, but DSB complexity-dependent manner following exposure to IR. Furthermore, ATR pathway also functioned more efficiently in ATM-proficient cells to block G2 to M transition at early period of particle radiation exposure. Accordingly, in contrast to ATM inhibitor, ATR inhibitor had a more effective radiosensitizing effect on survival fraction following heavy ion beams as compared with X ray radiation. Taken together, our results reveal that the complexity of DSBs is a crucial factor for the activation of ATR pathway for G2/M checkpoint regulation, and ATM-dependent end resection is not essential for the activation.     

DNA-PK: A dynamic enzyme in a versatile DSB repair pathway

DNA double stranded breaks (DSBs) are the most cytoxic DNA lesion as the inability to properly repair them can lead to genomic instability and tumorigenesis. The prominent DSB repair pathway in humans is non-homologous end-joining (NHEJ). In the simplest sense, NHEJ mediates the direct re-ligation of the broken DNA molecule. However, NHEJ is a complex and versatile process that can repair DSBs with a variety of damages and ends via the utilization of a significant number of proteins. In this review we will describe the important factors and mechanisms modulating NHEJ with emphasis given to the versatility of this repair process and the DNA-PK complex.     

The LC8-RavP ensemble Structure Evinces A Role for LC8 in Regulating Lyssavirus Polymerase Functionality

The rabies and Ebola viruses recruit the highly conserved host protein LC8 for their own reproductive success. In vivo knockouts of the LC8 recognition motif within the rabies virus phosphoprotein (RavP) result in completely nonlethal viral infections. In this work, we examine the molecular role LC8 plays in viral lethality. We show that RavP and LC8 colocalize in rabies infected cells, and that LC8 interactions are essential for efficient viral polymerase functionality. NMR, SAXS, and molecular modeling demonstrate that LC8 binding to a disordered linker adjacent to an endogenous dimerization domain results in restrictions in RavP domain orientations. The resulting ensemble structure of RavP-LC8 tetrameric complex is similar to that of a related virus phosphoprotein that does not bind LC8, suggesting that with RavP, LC8 binding acts as a switch to induce a more active conformation. The high conservation of the LC8 motif in Lyssavirus phosphoproteins and its presence in other analogous proteins such as the Ebola virus VP35 evinces a broader purpose for LC8 in regulating downstream phosphoprotein functions vital for viral replication.     

Characterization of a potent dominant negative mutant variant of Rad51 in Ustilago maydis

Rad51 serves to maintain and protect integrity of the genome through its actions in DNA repair and replication fork protection. The active form of Rad51 is a nucleoprotein filament consisting of chains of protomer units arranged linearly along single-stranded DNA. In a mutant screen using Ustilago maydis as an experimental system we identified a novel variant of Rad51, in which an amino acid change near the protomer–protomer interaction interface confers a strong trans dominant inhibitory effect on resistance to DNA damaging agents and proficiency in homologous recombination. Modeling studies of the mutated residue D161Y suggested that steric interference with surrounding residues was the likely cause of the inhibitory effect. Changes of two nearby residues, predicted from the modeling to minimize steric clashes, mitigated the inhibition of DNA repair. Direct testing of purified Rad51^D161Y protein in defined biochemical reactions revealed it to be devoid of DNA-binding activity itself, but capable of interfering with Rad51^WT in formation and maintenance of nucleoprotein filaments on single-stranded DNA and in DNA strand exchange. Rad51^D161Y protein appears to be unable to self-associate in solution and defective in forming complexes with the U. maydis BRCA2 ortholog.     

ATMIN is required for the ATM-mediated signaling and recruitment of 53BP1 to DNA damage sites upon replication stress

Unresolved replication intermediates can block the progression of replication forks and become converted into DNA lesions, hence exacerbating genomic instability. The p53-binding protein 1 (53BP1) forms nuclear bodies at sites of unrepaired DNA lesions to shield these regions against erosion, in a manner dependent on the DNA damage kinase ATM. The molecular mechanism by which ATM is activated upon replicative stress to localize the 53BP1 protection complex is unknown. Here we show that the ATM-INteracting protein ATMIN (also known as ASCIZ) is partially required for 53BP1 localization upon replicative stress. Additionally, we demonstrate that ATM activation is impaired in cells lacking ATMIN and we define that ATMIN is required for initiating ATM signaling following replicative stress. Furthermore, loss of ATMIN leads to chromosomal segregation defects. Together these data reveal that chromatin integrity depends on ATMIN upon exposure to replication-induced stress.     

RNA-directed repair of DNA double-strand breaks

DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, which if unrepaired or repaired incorrectly can cause cell death or genome instability that may lead to cancer. To counteract these adverse consequences, eukaryotes have evolved a highly orchestrated mechanism to repair DSBs, namely DNA-damage-response (DDR). DDR, as defined specifically in relation to DSBs, consists of multi-layered regulatory modes including DNA damage sensors, transducers and effectors, through which DSBs are sensed and then repaired via DNAprotein interactions. Unexpectedly, recent studies have revealed a direct role of RNA in the repair of DSBs, including DSB-induced small RNA (diRNA)-directed and RNA-templated DNA repair. Here, we summarize the recent discoveries of RNA-mediated regulation of DSB repair and discuss the potential impact of these novel RNA components of the DSB repair pathway on genomic stability and plasticity.     

LAMMER kinase contributes to genome stability in Ustilago maydis

Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1^Q488*) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1^Q488* rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1^Q488* mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1^Q488* mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis.     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

Identification of critical regions within the TIR domain of IL-1 receptor type I

Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these residues was investigated by site-directed mutagenesis and a functional luciferase assay reflecting NF-κB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL-1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the αC′ helix (Q469-E473 in mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.