The cellular responses to DNA damage

The ability to survive spontaneous and induced DNA damage, and to minimize the number of heritable mutations that this causes, is essential to the maintenance of genome integrity for all organisms. Early studies on model eukaryotes focused on genes acting in defined DNA repair pathways. More recent work with the budding and fission yeasts and mammalian cells has started to integrate the DNA damage response with cell physiology and the cell cycle.     

The role of chromatin proteins in DNA damage recognition and repair Mini-review

The structure of chromatin is the major factor determining the rate and efficiency of DNA repair. Chromatin remodeling events such as rearrangement of nucleosomes and higher order chromatin structures are indispensable features of repair processes. During the last decade numerous chromatin proteins have been identified that preferentially bind to different types of DNA damage. The HMGB proteins, which preferentially interact with DNA intrastrand crosslinks induced by cisplatin, are the archetypal example of such proteins. Several hypothetical models have been proposed describing the role of such damage-binding chromatin proteins. The damage shielding model postulates that binding of chromatin proteins to damaged DNA might disturb damage recognition by repair factors and impair its removal. Alternatively, the damage-recognition/signaling model proposes that the binding of specific chromatin proteins to damaged DNA could serve as a hallmark to be recognized by repair proteins. Additionally, the binding of specific chromatin proteins to damaged DNA could induce chromatin remodeling at the damage site and indirectly affect its repair. This paper aims to critically review current experimental data in relation to such possible roles of chromatin proteins.     

DNA damage responses to oxidative stress

The DNA damage response is a hierarchical process. DNA damage is detected by sensor proteins such as the MRN complex that transmit the information to transducer proteins such as ATM and ATR, which control the damage response through the phosphorylation of effector proteins. The extent of the DNA damage determines cell fate: cell cycle arrest and DNA repair or the activation of apoptotic pathways. In aerobic cells, reactive oxygen species (ROS) are generated as a by-product of normal mitochondrial activity. If not properly controlled, ROS can cause severe damage to cellular macromolecules, especially the DNA. We describe here some of the cellular responses to alterations in the cellular redox state during hypoxia or oxidative stress. Oxidative damage in DNA is repaired primarily via the base excision repair (BER) pathway which appears to be the simplest of the three excision repair pathways. To allow time for DNA repair, the cells activate their cell cycle checkpoints, leading to cell cycle arrest and preventing the replication of damage and defective DNA.     

DNA damage responses in the prostate

Comment on: Zhewei Z, et al. Cell Cycle 2011; 10:3545-53.     

The role of DNA damage response proteins at telomeres--an "integrative" model

Telomeres are specialized structures at chromosome ends which play the key role in chromosomal end protection. There is increasing evidence that many DNA damage response proteins are involved in telomere maintenance. For example, cells defective in DNA double strand break repair proteins including Ku, DNA-PKcs, RAD51D and the MRN (MRE11/RAD51/NBS1) complex show loss of telomere capping function. Similarly, mouse and human cells defective in ataxia telangiectasia mutated (ATM) have defective telomeres. A total of 14 mammalian DNA damage response proteins have, so far, been implicated in telomere maintenance. Recent studies indicate that three more proteins, namely BRCA1, hRad9 and PARP1 are involved in telomere maintenance. The involvement of a wide range of DNA damage response proteins at telomeres raises an important question: do telomere maintenance mechanisms constitute an integral part of DNA damage response machinery? A model termed the "integrative" model is proposed here to argue in favour of telomere maintenance being an integral part of DNA damage response. The "integrative" model is supported by the observation that a telomeric protein, TRF2, is not confined to its local telomeric environment but it migrates to the sites of DNA breakage following exposure of cells to ionizing radiation. Furthermore, even if telomeres are maintained in a non-canonical way, as in the case of Drosophila, DNA damage response proteins are still involved in telomere maintenance suggesting integration of telomere maintenance mechanisms into the DNA damage response network.     

Spermatocyte responses in vitro to induced DNA damage

Spermatocytes normally sustain many meiotically induced double-strand DNA breaks (DSBs) early in meiotic prophase; in autosomal chromatin, these are repaired by initiation of meiotic homologous-recombination processes. Little is known about how spermatocytes respond to environmentally induced DNA damage after recombination-related DSBs have been repaired. The experiments described here tested the hypothesis that, even though actively completing meiotic recombination, pachytene spermatocytes cultured in the absence of testicular somatic cells initiate appropriate chromatin remodeling and cell-cycle responses to environmentally induced DNA damage. Two DNA-damaging agents were employed for in vitro treatment of pachytene spermatocytes: gamma-irradiation and etoposide, a topoisomerase II (TOP2) inhibitor that results in persistent unligated DSBs. Chromatin modifications associated with DSBs were monitored after exposure by labeling surface-spread chromatin with antibodies against RAD51 (which recognizes DSBs) and the phosphorylated variant of histone H2AFX (herein designated by its commonly used symbol, H2AX), gammaH2AX (which modifies chromatin associated with DSBs). Both gammaH2AX and RAD51 were rapidly recruited to irradiation- or etoposide-damaged chromatin. These chromatin modifications imply that spermatocytes recruit active DNA damage responses, even after recombination is substantially completed. Furthermore, irradiation-induced DNA damage inhibited okadaic acid-induced progression of spermatocytes from meiotic prophase to metaphase I (MI), implying efficacy of DNA damage checkpoint mechanisms. Apoptotic responses of spermatocytes with DNA damage differed, with an increase in frequency of early apoptotic spermatocytes after etoposide treatment, but not following irradiation. Taken together, these results demonstrate modification of pachytene spermatocyte chromatin and inhibition of meiotic progress after DNA damage by mechanisms that may ensure gametic genetic integrity.     

The evolution of ATPase activity in SMC proteins

The SMC (structural maintenance of chromosomes) proteins are a highly conserved and ubiquitous family of ATPases, found in nearly all living organisms examined, where they play crucial roles in transmission of the hereditary material. However, the extent to which efficient ATP hydrolysis is required for SMC function has been a matter of some debate. Here we investigate the potential functional significance of ATP binding and hydrolysis in different eukaryotic SMC proteins, both by comparing the conservation of conserved ATPase motifs and by exploring potential coevolution between associated domains. In this way, we have been able to account for the reduced requirement for ATPase activity in cohesin's SMC3 and demonstrate the greater apparent conservation requirements for such activity in condensin SMC proteins. Finally, we explore possible interactions between the SMC and non-SMC components of the condensin complex that are required for full condensin activity and may modulate ATPase activity in the holocomplex.     

The role of arginine methylation in the DNA damage response

Post-translational modifications are well-known modulators of DNA damage signaling and epigenetic gene expression. Protein arginine methylation is a covalent modification that results in the addition of methyl groups to the nitrogen atoms of the arginine side chains and is catalyzed by a family of protein arginine methyltransferases (PRMTs). In the past, arginine methylation was mainly observed on abundant proteins such as RNA-binding proteins and histones, but recent advances have revealed a plethora of arginine methylated proteins implicated in a variety of cellular processes including RNA metabolism, epigenetic regulation and DNA repair pathways. Herein, we discuss these recent advances, focusing on the role of PRMTs in DNA damage signaling and its importance for maintaining genomic stability.     

The ubiquitous role of ubiquitin in the DNA damage response

Protein ubiquitylation has emerged as an important regulatory mechanism that impacts almost every aspect of the DNA damage response. In this review, we discuss how DNA repair and checkpoint pathways utilize the diversity offered by the ubiquitin conjugation system to modulate the response to genotoxic lesions in space and time. In particular, we will highlight recent work done on the regulation of DNA double-strand breaks signalling and repair by the RNF8/RNF168 E3 ubiquitin ligases, the Fanconi anemia pathway and the role of protein degradation in the enforcement and termination of checkpoint signalling. We also discuss the various functions of deubiquitylating enzymes in these processes along with potential avenues for exploiting the ubiquitin conjugation/deconjugation system for therapeutic purposes.     

Nuclear reorganization of DNA mismatch repair proteins in response to DNA damage

The DNA mismatch repair (MMR) system is highly conserved and vital for preserving genomic integrity. Current mechanistic models for MMR are mainly derived from in vitro assays including reconstitution of strand-specific MMR and DNA binding assays using short oligonucleotides. However, fundamental questions regarding the mechanism and regulation in the context of cellular DNA replication remain. Using synchronized populations of HeLa cells we demonstrated that hMSH2, hMLH1 and PCNA localize to the chromatin during S-phase, and accumulate to a greater extent in cells treated with a DNA alkylating agent. In addition, using small interfering RNA to deplete hMSH2, we demonstrated that hMLH1 localization to the chromatin is hMSH2-dependent. hMSH2/hMLH1/PCNA proteins, when associated with the chromatin, form a complex that is greatly enhanced by DNA damage. The DNA damage caused by high doses of alkylating agents leads to a G₂ arrest after only one round of replication. In these G₂-arrested cells, an hMSH2/hMLH1 complex persists on chromatin, however, PCNA is no longer in the complex. Cells treated with a lower dose of alkylating agent require two rounds of replication before cells arrest in G₂. In the first S-phase, the MMR proteins form a complex with PCNA, however, during the second S-phase PCNA is missing from that complex. The distinction between these complexes may suggest separate functions for the MMR proteins in damage repair and signaling. Additionally, using confocal immunofluorescence, we observed a population of hMSH6 that localized to the nucleolus. This population is significantly reduced after DNA damage suggesting that the protein is shuttled out of the nucleolus in response to damage. In contrast, hMLH1 is excluded from the nucleolus at all times. Thus, the nucleolus may act to segregate a population of hMSH2–hMSH6 from hMLH1–hPMS2 such that, in the absence of DNA damage, an inappropriate response is not invoked.     

The interplay of Fanconi anemia proteins in the DNA damage response

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by chromosome instability and cancer predisposition. At least 11 complementation groups for FA have been identified, and eight FA genes have been cloned. Interestingly, the eight known FA proteins cooperate in a common pathway leading to the interaction of monoubiquitinated FANCD2 and BRCA2 in damaged chromatin. Disruption of this pathway results in the clinical and cellular abnormalities common to all FA subtypes. This review will examine the interaction of the cloned FA proteins with each other and with other DNA damage response proteins (i.e., ATM, ATR, and NBS1). Also, somatic (acquired) disruption of the FA pathway in human tumors appears to account for their chromosome instability and crosslinker hypersensitivity.     

The role of metals in site-specific DNA damage with reference to carcinogenesis

We reviewed the mechanism of oxidative DNA damage with reference to metal carcinogenesis and metal-mediated chemical carcinogenesis. On the basis of the finding that chromium (VI) induced oxidative DNA damage in the presence of hydrogen peroxide (H2O2), we proposed the hypothesis that endogenous reactive oxygen species play a role in metal carcinogenesis. Since then, we have reported that various metal compounds, such as cobalt, nickel, and ferric nitrilotriacetate, directly cause site-specific DNA damage in the presence of H2O2. We also found that carcinogenic metals could cause DNA damage through indirect mechanisms. Certain nickel compounds induced oxidative DNA damage in rat lungs through inflammation. Endogenous metals, copper and iron, catalyzed ROS generation from various organic carcinogens, resulting in oxidative DNA damage. Polynuclear compounds, such as 4-aminobiphenyl and heterocyclic amines, appear to induce cancer mainly through DNA adduct formation, although their N-hydroxy and nitroso metabolites can also cause oxidative DNA damage. On the other hand, mononuclear compounds, such as benzene metabolites, caffeic acid, and o-toluidine, should express their carcionogenicity through oxidative DNA damage. Metabolites of certain carcinogens efficiently caused oxidative DNA damage by forming NADH-dependent redox cycles. These findings suggest that metal-mediated oxidative DNA damage plays important roles in chemical carcinogenesis.     

DNA damage responses after exposure to DNA-based products

BACKGROUND: The development of DNA-based therapies holds great promise for the treatment of diseases that remain difficult to manage using conventional pharmaceuticals. Whilst there are considerable data regarding chemical-induced DNA damage, there are limited reports published studying the potential of exogenous DNA to damage genomic DNA. METHODS: To investigate this problem, the differential gene expression (DGE) of DNA repair genes was examined to identify biomarkers, based on the hypothesis that DNA damage, including double-strand breaks (DSBs) and insertional mutagenesis, would be expected to induce biological pathways associated with repair. Human HepG2 cells were exposed to the chemical genotoxins, etoposide (ETOP) and methylmethanesulphonate (MMS), as positive controls, or biological agents (i.e. exogenous DNA with and without the use of transfection complexes or via various viral vectors). Following transfection (6-72 h) the cells were harvested for RNA and DGE was determined by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The expression of genes involved in the repair of DSBs were significantly increased after treatment with ETOP (>4-fold) or MMS (>5-fold). Transfection using Effectene and ExGen 500 resulted in no significant changes; however, transfection with ExGen 500 resulted in an increase in the expression levels of GADD45 mRNA, consistent with global cellular stress. Viral vectors increased (3-6-fold) expression of genes associated with DSBs and cellular stress responses and, as expected, the effect was the most marked with the retroviral vector. CONCLUSIONS: The DGE profiles observed in HepG2 cells following transduction/transfection suggest that a subset of DNA repair genes may provide novel biomarkers to rapidly detect DNA damage induced by DNA products at the level of the genome, rather than at selected genes.     

RecQ helicases and cellular responses to DNA damage

The faithful replication of the genome is essential for the survival of all organisms. It is not surprising therefore that numerous mechanisms have evolved to ensure that duplication of the genome occurs with only minimal risk of mutation induction. One mechanism of genome destabilization is replication fork demise, which can occur when a translocating fork meets a lesion or adduct in the template. Indeed, the collapse of replication forks has been suggested to occur in every replicative cell cycle making this a potentially significant problem for all proliferating cells. The RecQ helicases, which are essential for the maintenance of genome stability, are thought to function during DNA replication. In particular, RecQ helicase mutants display replication defects and have phenotypes consistent with an inability to efficiently reinitiate replication following replication fork demise. Here, we review some current models for how replication fork repair might be effected, and discuss potential roles for RecQ helicases in this process.     

乳腺癌易感蛋白1在DNA损伤修复中的作用

人类乳腺癌易感基因1(breast cancer susceptibility gene 1,BRCA1)首先是在乳腺癌家族中发现的,是具有遗传倾向的乳腺癌和卵巢癌易感基因,其基因的突变与家族性乳腺癌及卵巢癌的发生有密切联系。BRCA1是一种抑癌基因,其基因产物可以参与维持基因组稳定性的多条细胞信号通路,例如DNA损伤诱导的细胞周期调控、DNA损伤修复、基因转录调节、细胞凋亡、泛素化等重要的细胞活动。本文就近几年来BRCA1在DNA损伤修复中的作用的研究进展作一综述,包括DNA损伤诱导的细胞周期检查点的激活和DNA损伤修复两方面。