The nucleotide excision repair pathway is required for UV-C-induced apoptosis in Caenorhabditis elegans

Ultraviolet (UV) radiation is a mutagen of major clinical importance in humans. UV-induced damage activates multiple signaling pathways, which initiate DNA repair, cell cycle arrest and apoptosis. To better understand these pathways, we studied the responses to UV-C light (254 nm) of germ cells in Caenorhabditis elegans. We found that UV activates the same cellular responses in worms as in mammalian cells. Both UV-induced apoptosis and cell cycle arrest were completely dependent on the p53 homolog CEP-1, the checkpoint proteins HUS-1 and CLK-2, and the checkpoint kinases CHK-2 and ATL-1 (the C. elegans homolog of ataxia telangiectasia and Rad3-related); ATM-1 (ataxia telangiectasia mutated-1) was also required, but only at low irradiation doses. Importantly, mutation of genes encoding nucleotide excision repair pathway components severely disrupted both apoptosis and cell cycle arrest, suggesting that these genes not only participate in repair, but also signal the presence of damage to downstream components of the UV response pathway that we delineate here. Our study suggests that whereas DNA damage response pathways are conserved in metazoans in their general outline, there is significant evolution in the relative importance of individual checkpoint genes in the response to specific types of DNA damage.     

Involvement of Nucleotide Excision and Mismatch Repair Mechanisms in Double Strand Break Repair

Living organisms are constantly threatened by environmental DNA-damaging agents, including UV and ionizing radiation (IR). Repair of various forms of DNA damage caused by IR is normally thought to follow lesion-specific repair pathways with distinct enzymatic machinery. DNA double strand break is one of the most serious kinds of damage induced by IR, which is repaired through double strand break (DSB) repair mechanisms, including homologous recombination (HR) and non-homologous end joining (NHEJ). However, recent studies have presented increasing evidence that various DNA repair pathways are not separated, but well interlinked. It has been suggested that non-DSB repair mechanisms, such as Nucleotide Excision Repair (NER), Mismatch Repair (MMR) and cell cycle regulation, are highly involved in DSB repairs. These findings revealed previously unrecognized roles of various non-DSB repair genes and indicated that a successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems. One of our recent studies found that suppressed expression of non-DSB repair genes, such as XPA, RPA and MLH1, influenced the yield of IR induced micronuclei formation and/or chromosome aberrations, suggesting that these genes are highly involved in DSB repair and DSB-related cell cycle arrest, which reveals new roles for these gene products in the DNA repair network. In this review, we summarize current progress on the function of non-DSB repair-related proteins, especially those that participate in NER and MMR pathways, and their influence on DSB repair. In addition, we present our developing view that the DSB repair mechanisms are more complex and are regulated by not only the well known HR/NHEJ pathways, but also a systematically coordinated cellular network.Key Words: Ionizing radiation (IR), DNA damage, DSB repair, NER, MMR and cell cycle.     

Ataxia telangiectasia-mutated dependent DNA damage checkpoint functions regulate gene expression in human fibroblasts

The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6, and 24 h after treatment with 1.5-Gy IR or sham treatment and were compared with those previously recognized in normal human fibroblasts. Under basal conditions, 160 genes or expressed sequence tags were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. On DNA damage, 1,091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1,811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth, and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison with normal cells. The reduced change in DNA damage response genes and the attenuated repression of cell cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells.     

Gamma-irradiation and doxorubicin treatment of normal human cells cause cell cycle arrest via different pathways

Ionizing radiation and doxorubicin both produce oxidative damage and double-strand breaks in DNA. Double-strand breaks and oxidative damage are highly toxic and cause cell cycle arrest, provoking DNA repair and apoptosis in cancer cell lines. To investigate the response of normal human cells to agents causing oxidative damage, we monitored alterations in gene expression in F65 normal human fibroblasts. Treatment with g-irradiation and doxorubicin altered the expression of 23 and 68 known genes, respectively, with no genes in common. Both agents altered the expression of genes involved in cell cycle arrest, and arrested the treated cells in G2/M phase 12 h after treatment. 24 h after g-irradiation, the percentage of G1 cells increased, whereas after doxorubicin treatment the percentage of G2/M cells remained constant for 24 h. Our results suggest that F65 cells respond differently to g-irradiation- and doxorubicin-induced DNA damage, probably using entirely different biochemical pathways.     

Escherichia coli Genes and Pathways Involved in Surviving Extreme Exposure to Ionizing Radiation

To further an improved understanding of the mechanisms used by bacterial cells to survive extreme exposure to ionizing radiation (IR), we broadly screened nonessential Escherichia coli genes for those involved in IR resistance by using transposon-directed insertion sequencing (TraDIS). Forty-six genes were identified, most of which become essential upon heavy IR exposure. Most of these were subjected to direct validation. The results reinforced the notion that survival after high doses of ionizing radiation does not depend on a single mechanism or process, but instead is multifaceted. Many identified genes affect either DNA repair or the cellular response to oxidative damage. However, contributions by genes involved in cell wall structure/function, cell division, and intermediary metabolism were also evident. About half of the identified genes have not previously been associated with IR resistance or recovery from IR exposure, including eight genes of unknown function.     

Locations of mouse DNA damage response and repair loci, and cancer risk modifiers

An outline is presented of an electronically accessible database that compares the locations of mouse genes involved in DNA repair, apoptosis, cell cycle and signal transduction with those of known cancer risk modifier genes. The database has a primary but not exclusive focus on modifiers of ionizing radiation (IR) cancer risk and genes involved in IR-induced DNA damage responses. The database () provides a useful tool for assessing the role of DNA damage response genes in cancer predisposition.     

Analysis of LexA binding sites and transcriptomics in response to genotoxic stress in Leptospira interrogans

We determined the effects of DNA damage caused by ultraviolet radiation on gene expression in Leptospira interrogans using DNA microarrays. These data were integrated with DNA binding in vivo of LexA1, a regulator of the DNA damage response, assessed by chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq). In response to DNA damage, Leptospira induced expression of genes involved in DNA metabolism, in mobile genetic elements and defective prophages. The DNA repair genes involved in removal of photo-damage (e.g. nucleotide excision repair uvrABC, recombinases recBCD and resolvases ruvABC) were not induced. Genes involved in various metabolic pathways were down regulated, including genes involved in cell growth, RNA metabolism and the tricarboxylic acid cycle. From ChIP-seq data, we observed 24 LexA1 binding sites located throughout chromosome 1 and one binding site in chromosome 2. Expression of many, but not all, genes near those sites was increased following DNA damage. Binding sites were found as far as 550 bp upstream from the start codon, or 1 kb into the coding sequence. Our findings indicate that there is a shift in gene expression following DNA damage that represses genes involved in cell growth and virulence, and induces genes involved in mutagenesis and recombination.     

Local false discovery rate facilitates comparison of different microarray experiments

The local false discovery rate (LFDR) estimates the probability of falsely identifying specific genes with changes in expression. In computer simulations, LFDR <10% successfully identified genes with changes in expression, while LFDR >90% identified genes without changes. We used LFDR to compare different microarray experiments quantitatively: (i) Venn diagrams of genes with and without changes in expression, (ii) scatter plots of the genes, (iii) correlation coefficients in the scatter plots and (iv) distributions of gene function. To illustrate, we compared three methods for pre-processing microarray data. Correlations between methods were high (r = 0.84–0.92). However, responses were often different in magnitude, and sometimes discordant, even though the methods used the same raw data. LFDR complements functional assessments like gene set enrichment analysis. To illustrate, we compared responses to ultraviolet radiation (UV), ionizing radiation (IR) and tobacco smoke. Compared to unresponsive genes, genes responsive to both UV and IR were enriched for cell cycle, mitosis, and DNA repair functions. Genes responsive to UV but not IR were depleted for cell adhesion functions. Genes responsive to tobacco smoke were enriched for detoxification functions. Thus, LFDR reveals differences and similarities among experiments.     

Genomic instability and cancer: networks involved in response to DNA damage

A new approach to cancer and new methods in examining rare human chromosome breakage syndromes have brought to light complex interactions between different pathways involved in damage response, cell cycle checkpoint control and DNA repair. The genes affected in these different syndromes are involved in networks of processes that respond to DNA damage and prevent chromosomal aberrations during the cell cycle. The genes involved include the ATM, ATR, FA-associated genes, NBS1 and the cancer susceptibility genes BRCA1 and BRCA2. Chromosomal instability is a common feature of many human cancers and most of the instability syndromes, characterized by sensitivity to different types of DNA damage, also show increased cancer susceptibility. Better understanding of these syndromes and their links with familial cancer provide new insight into associations between defects in DNA damage response, cell cycle control, DNA repair and cancer. Understanding the damage response repair networks that these studies are revealing will have important implications for the development of cancer management and treatment.     

Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints.

ATR, a phosphatidylinositol kinase-related protein homologous to ataxia telangiectasia mutated (ATM), is important for the survival of human cells following many forms of DNA damage. Expression of a kinase-inactive allele of ATR (ATRkd) in human fibroblasts causes increased sensitivity to ionizing radiation (IR), cis-platinum and methyl methanesulfonate, but only slight UV radiation sensitivity. ATRkd overexpression abrogates the G2/M arrest after exposure to IR, and overexpression of wild-type ATR complements the radioresistant DNA synthesis phenotype of cells lacking ATM, suggesting a potential functional overlap between these proteins. ATRkd overexpression also causes increased sensitivity to hydroxyurea that is associated with microtubule-mediated nuclear abnormalities. These observations are consistent with uncoupling of certain mitotic events from the completion of S-phase. Thus, ATR is an important component of multiple DNA damage response pathways and may be involved in the DNA replication (S/M) checkpoint.     

Rad3 and Sty1 function in Schizosaccharomyces pombe: an integrated response to DNA damage and environmental stress?

In Schizosaccharomyces pombe , the Ataxia Telangiectasia-mutated (Atm)/Atm and Rad 3 Related (Atr) homologue Rad3 is an essential regulator of the response to DNA damage and stalled replication forks. Rad3 activates the downstream kinases Chk1 and Cds1. These kinases in turn inhibit cell cycle progression by mediating Cdc2 phosphorylation. Studies in both yeast and mammalian cells suggest additional roles for Rad3 in regulating cellular responses to environmental stress. In S. pombe , cellular responses to various environmental stresses are regulated primarily through the stress-activated MAP kinase p38 homologue Sty1. An important function of Sty1 is to drive cells rapidly through mitosis by facilitating the accumulation of Cdc25. Interestingly, Sty1 is activated simultaneously with Rad3 following exposure to UV radiation or ionizing radiation (IR). Similarly, exposure to environmental stresses induces the expression of rad3 ⁺, cds1 ⁺ and other checkpoint regulator genes. It is currently unclear how the pathways regulated by Sty1 and Rad3 and their opposing effects on mitosis are integrated. Recent studies suggest that Sty1 and Rad3 function together to regulate the expression of several stress response genes following exposure to IR. In this review, we discuss current knowledge on the interaction of Rad3/Atm and Sty1/p38 in regulating cellular responses to environmental stress and DNA damage.