Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B1 treatment was assayed in strains defective in nucleotide excision repair (NER) (rad14), postreplication repair (PRR) (rad6, rad18, mms2, and rad5), homologous recombinational repair (HRR) (rad51 and rad54), base excision repair (BER) (apn1 apn2), nonhomologous end-joining (NHEJ) (yku70), mismatch repair (MMR) (pms1), translesion synthesis (TLS) (rev3), and checkpoints (mec1-1, mec1-1 rad53, rad9, and rad17). Together our data suggest the involvement of homologous recombination and nucleotide excision repair, postreplication repair, and checkpoints in the repair and/or tolerance of AFB1-induced DNA damage in the yeast model. Rev3 appears to mediate AFB1-induced mutagenesis when error-free pathways are compromised. The results further suggest unique roles for Rad5 and abasic endonuclease-dependent DNA intermediates in regulating AFB1-induced mutagenicity.
DNA interstrand cross-links (ICLs) present a major challenge to cells, preventing separation of the two strands of duplex DNA and blocking major chromosome transactions, including transcription and replication. Due to the complexity of removing this form of DNA damage, no single DNA repair pathway has been shown to be capable of eradicating ICLs. In eukaryotes, ICL repair is a complex process, principally because several repair pathways compete for ICL repair intermediates in a strictly cell cycle-dependent manner. Yeast cells require a combination of nucleotide excision repair, homologous recombination repair and postreplication repair/translesion DNA synthesis to remove ICLs. There are also a number of additional ICL repair factors originally identified in the budding yeast Saccharomyces cerevisiae, called Pso1 though 10, of which Pso2 has an apparently dedicated role in ICL repair. Mammalian cells respond to ICLs by a complex network guided by factors mutated in the inherited cancer-prone disorder Fanconi anemia (FA). Although enormous progress has been made over recent years in identifying and characterizing FA factors as well as in elucidating certain aspects of the biology of FA, the mechanistic details of the ICL repair defects in FA patients remain unknown. Dissection of the FA DNA damage response pathway has, in part, been limited by the absence of FA-like pathways in highly tractable model organisms, such as yeast. Although S. cerevisiae possesses putative homologs of the FA factors FANCM, FANCJ and FANCP (Mph1, Chl1 and Slx4, respectively) as well as of the FANCM-associated proteins MHF1 and MHF2 (Mhf1 and Mhf2), the corresponding mutants display no significant increase in sensitivity to ICLs. Nevertheless, we and others have recently shown that these FA homologs, along with several other factors, control an ICL repair pathway, which has an overlapping or redundant role with a Pso2-controlled pathway. This pathway acts in S-phase and serves to prevent ICL-stalled replication forks from collapsing into DNA double-strand breaks.
Fanconi anemia; DNA interstrand cross-link repair; S-phase; Saccharomyces cerevisiae
Plants use the energy in sunlight for photosynthesis, but as a consequence are exposed to the toxic effect of UV radiation especially on DNA. The UV-induced lesions on DNA affect both transcription and replication and can also have mutagenic consequences. Here we investigated the regulation and the function of the recently described CUL4-DDB1-DDB2 E3 ligase in the maintenance of genome integrity upon UV-stress using the model plant Arabidopsis. Physiological, biochemical, and genetic evidences indicate that this protein complex is involved in global genome repair (GGR) of UV-induced DNA lesions. Moreover, we provide evidences for crosstalks between GGR, the plant-specific photo reactivation pathway and the RAD1-RAD10 endonucleases upon UV exposure. Finally, we report that DDB2 degradation upon UV stress depends not only on CUL4, but also on the checkpoint protein kinase Ataxia telangiectasia and Rad3-related (ATR). Interestingly, we found that DDB1A shuttles from the cytoplasm to the nucleus in an ATR-dependent manner, highlighting an upstream level of control and a novel mechanism of regulation of this E3 ligase.
Recent research revealed strong links between Cullin4 (CUL4)–based cullin-RING ubiquitin ligases (CRLs) and chromatin biology, including DNA replication and DNA repair. During Nucleotide Excision Repair (NER), CUL4 together with DDB1 (DNA Damage Binding protein 1) ubiquitylate an increasingly large number of substrates, including components of the NER machinery as well as various histone proteins. In contrast to mammals, plants have an efficient DNA repair pathway, mediated by photolyases that reverse UV lesions in presence of visible light, without DNA excision. Thus, it is believed that in aerial plant tissues this DNA repair pathway is predominant. In the present work we used the model plant Arabidopsis to investigate the role of CUL4-DDB1ADDB2 in global genome repair at the level of a whole organism. Using a genetic approach, we highlighted the existence of cooperative roles of different DNA repair processes that all together contribute to maintain genome integrity upon exposure to UV. Moreover, we report that DDB2 turnover not only depends on CUL4-DDB1A, but also on the ATR checkpoint protein kinase. Strikingly, we demonstrated that Arabidopsis DDB1A shuttles from the cytoplasm to the nucleus after UV stress in an ATR-dependent manner, illustrating a novel level of regulation of this class of CRL.
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.
Ionizing radiation (IR); DNA damage; DSB repair; NER; MMR and cell cycle.
The base excision repair (BER) that repairs oxidative damage is upregulated as an adaptive response in maintaining tumorigenesis of RAS-transformed cancer cells.
The Cut homeobox 1 (CUX1) gene is a target of loss-of-heterozygosity in many cancers, yet elevated CUX1 expression is frequently observed and is associated with shorter disease-free survival. The dual role of CUX1 in cancer is illustrated by the fact that most cell lines with CUX1 LOH display amplification of the remaining allele, suggesting that decreased CUX1 expression facilitates tumor development while increased CUX1 expression is needed in tumorigenic cells. Indeed, CUX1 was found in a genome-wide RNAi screen to identify synthetic lethal interactions with oncogenic RAS. Here we show that CUX1 functions in base excision repair as an ancillary factor for the 8-oxoG-DNA glycosylase, OGG1. Single cell gel electrophoresis (comet assay) reveals that Cux1+/− MEFs are haploinsufficient for the repair of oxidative DNA damage, whereas elevated CUX1 levels accelerate DNA repair. In vitro base excision repair assays with purified components demonstrate that CUX1 directly stimulates OGG1's enzymatic activity. Elevated reactive oxygen species (ROS) levels in cells with sustained RAS pathway activation can cause cellular senescence. We show that elevated expression of either CUX1 or OGG1 prevents RAS-induced senescence in primary cells, and that CUX1 knockdown is synthetic lethal with oncogenic RAS in human cancer cells. Elevated CUX1 expression in a transgenic mouse model enables the emergence of mammary tumors with spontaneous activating Kras mutations. We confirmed cooperation between KrasG12V and CUX1 in a lung tumor model. Cancer cells can overcome the antiproliferative effects of excessive DNA damage by inactivating a DNA damage response pathway such as ATM or p53 signaling. Our findings reveal an alternate mechanism to allow sustained proliferation in RAS-transformed cells through increased DNA base excision repair capability. The heightened dependency of RAS-transformed cells on base excision repair may provide a therapeutic window that could be exploited with drugs that specifically target this pathway.
In the context of tumor development and progression, mutations are believed to accumulate owing to compromised DNA repair. Such mutations promote oncogenic growth. Yet cancer cells also need to sustain a certain level of DNA repair in order to replicate their DNA and successfully proliferate. Here we show that cancer cells that harbor an activated RAS oncogene exhibit heightened DNA repair capability, specifically in the base excision repair (BER) pathway that repairs oxidative DNA damage. RAS oncogenes alone do not transform primary cells but rather cause their senescence—that is, they stop dividing. As such, cellular senescence in this context is proposed to function as a tumor-suppressive mechanism. We show that CUX1, a protein that accelerates oxidative DNA damage repair, prevents cells from senescing and enables proliferation in the presence of a RAS oncogene. Consistent with this, RAS-induced senescence is also prevented by ectopic expression of OGG1, the DNA glycosylase that removes 8-oxoguanine, the most abundant oxidized base. Strikingly, CUX1 expression in transgenic mice enables the emergence of tumors with spontaneous activating Kras mutations. Conversely, knockdown of CUX1 is synthetic lethal for RAS-transformed cells, thereby revealing a potential Achilles' heel of these cancer cells. Overall, the work provides insight into understanding the role of DNA repair in cancer progression, showing that while DNA damage-induced mutations promote tumorigenesis, sustained RAS-dependent tumorigenesis requires suppression of DNA damage. The heightened dependency of RAS-transformed cells on base excision repair may provide a therapeutic window that could be exploited with drugs that specifically target this pathway.
During affinity maturation, genomic integrity is maintained through specific targeting of DNA mutations. The DNA damage sensor PARP-1 helps determine whether a DNA lesion results in faithful or mutagenic repair.
Genetic variation at immunoglobulin (Ig) gene variable regions in B-cells is created through a multi-step process involving deamination of cytosine bases by activation-induced cytidine deaminase (AID) and their subsequent mutagenic repair. To protect the genome from dangerous, potentially oncogenic effects of off-target mutations, both AID activity and mutagenic repair are targeted specifically to the Ig genes. However, the mechanisms of targeting are unknown and recent data have highlighted the role of regulating mutagenic repair to limit the accumulation of somatic mutations resulting from the more widely distributed AID-induced lesions to the Ig genes. Here we investigated the role of the DNA damage sensor poly-(ADPribose)-polymerase-1 (PARP-1) in the repair of AID-induced DNA lesions. We show through sequencing of the diversifying Ig genes in PARP-1−/− DT40 B-cells that PARP-1 deficiency results in a marked reduction in gene conversion events and enhanced high-fidelity repair of AID-induced lesions at both Ig heavy and light chains. To further characterize the role of PARP-1 in the mutagenic repair of AID-induced lesions, we performed functional analyses comparing the role of engineered PARP-1 variants in high-fidelity repair of DNA damage induced by methyl methane sulfonate (MMS) and the mutagenic repair of lesions at the Ig genes induced by AID. This revealed a requirement for the previously uncharacterized BRCT domain of PARP-1 to reconstitute both gene conversion and a normal rate of somatic mutation at Ig genes, while being dispensable for the high-fidelity base excision repair. From these data we conclude that the BRCT domain of PARP-1 is required to initiate a significant proportion of the mutagenic repair specific to diversifying antibody genes. This role is distinct from the known roles of PARP-1 in high-fidelity DNA repair, suggesting that the PARP-1 BRCT domain has a specialized role in assembling mutagenic DNA repair complexes involved in antibody diversification.
To produce a limitless diversity of antibodies within the constraints of a finite genome, activated B cells introduce random mutations into antibody genes through a process of targeted DNA damage and subsequent mutagenic repair. At the same time, the rest of the genome must be protected from mutagenesis to prevent off-target mutations which can lead to the development of lymphoma or leukemia. How antibody genes are specifically targeted is still largely unknown. A potential player in this process is the DNA-damage-sensing enzyme PARP-1, which recruits DNA repair enzymes to sites of damage. Using a chicken B cell lymphoma cell line because it has only a single PARP isoform and constitutively mutates its antibody genes, we compared the types of mutations accumulated in PARP-1−/− cells to wild type. We found that in cells lacking PARP-1, the major pathway of mutagenic repair was disrupted and fewer mutations than normal were introduced into their antibody genes. To identify what might be important for mutagenesis, we tested different factors for their ability to rescue this mutagenic deficiency and found a role for the BRCT (BRCA1 C-terminal) domain of PARP-1, a consensus protein domain known to be involved in directing protein-protein interactions. Our evidence suggests that PARP-1 may be interacting with another hypothetical protein via its BRCT domain that is required for the mutagenic rather than faithful repair of DNA lesions in the antibody genes.
DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM).
We identify that SVM techniques are capable of identifying portions of DNA repair protein datasets without admitting false positives; at low levels of false positive tolerance, homology can also identify and classify proteins with good performance. Secondary structure information provides improved performance compared to using primary structure alone. Furthermore, we observe that machine learning methods incorporating homology information perform best when data is filtered by some clustering technique. Analysis by applying these methodologies to the scanning of multiple vertebrate genomes confirms a positive correlation between the size of a genome and the number of DNA repair protein transcripts it is likely to contain, and simultaneously suggests that all organisms have a non-zero minimum number of repair genes. In addition, the scan result clusters several organisms' repair abilities in an evolutionarily consistent fashion. Analysis also identifies several functionally unconfirmed proteins that are highly likely to be involved in the repair process. A new web service, INTREPED, has been made available for the immediate search and annotation of DNA repair proteins in newly sequenced genomes.
Despite complexity due to a multitude of repair pathways, combinations of sequence, structure, and homology with Support Vector Machines offer good methods in addition to existing homology searches for DNA repair protein identification and functional annotation. Most importantly, this study has uncovered relationships between the size of a genome and a genome's available repair repetoire, and offers a number of new predictions as well as a prediction service, both which reduce the search time and cost for novel repair genes and proteins.
Organisms like Dictyostelium discoideum, often referred to as DNA damage “extremophiles”, can survive exposure to extremely high doses of radiation and DNA crosslinking agents. These agents form highly toxic DNA crosslinks that cause extensive DNA damage. However, little is known about how Dictyostelium and the other “extremophiles” can tolerate and repair such large numbers of DNA crosslinks. Here we describe a comprehensive genetic analysis of crosslink repair in Dictyostelium discoideum. We analyse three gene groups that are crucial for a replication-coupled repair process that removes DNA crosslinks in higher eukarya: The Fanconi anaemia pathway (FA), translesion synthesis (TLS), and nucleotide excision repair. Gene disruption studies unexpectedly reveal that the FA genes and the TLS enzyme Rev3 play minor roles in tolerance to crosslinks in Dictyostelium. However, disruption of the Xpf nuclease subcomponent results in striking hypersensitivity to crosslinks. Genetic interaction studies reveal that although Xpf functions with FA and TLS gene products, most Xpf mediated repair is independent of these two gene groups. These results suggest that Dictyostelium utilises a distinct Xpf nuclease-mediated repair process to remove crosslinked DNA. Other DNA damage–resistant organisms and chemoresistant cancer cells might adopt a similar strategy to develop resistance to DNA crosslinking agents.
Organisms are constantly exposed to environmental and endogenous molecules that chemically modify the DNA in their genomes. A particularly pernicious chemical modification is when the two strands of DNA are crosslinked. These crosslinks must be removed so that genomes can be copied, and the damage caused by their persistence is often exploited in cancer chemotherapy. It is also no surprise that all organisms have developed effective means to remove these lesions, and work in prokaryotes and eukaryotes has shown that crosslinks are removed by the concerted action of certain DNA repair pathways. Whilst the obvious route of accumulating crosslinks is by exposure to anti-cancer drugs, these lesions may also arise spontaneously in DNA. This could be why inherited inactivation of one of the crosslink repair pathways results in the catastrophic human illness Fanconi anaemia. Here we determine how the social amoeba Dictyostelium discoideum, an organism that is unusually resistant to DNA-damaging agents, removes crosslinks. Our results indicate that this organism has evolved a distinct strategy to remove these lesions. More specifically, we discover that a particular nuclease subcomponent removes the crosslinks by a distinct repair process. We postulate that this strategy to remove crosslinks could be used by other DNA damage–resistant organisms and also by cancer cells that have developed resistance to chemotherapy.
Eukaryotic cells possess several mechanisms to protect the integrity of their DNA against damage. These include cell-cycle checkpoints, DNA-repair pathways, and also a distinct DNA damage–tolerance system that allows recovery of replication forks blocked at sites of DNA damage. In both humans and yeast, lesion bypass and restart of DNA synthesis can occur through an error-prone pathway activated following mono-ubiquitination of proliferating cell nuclear antigen (PCNA), a protein found at sites of replication, and recruitment of specialized translesion synthesis polymerases. In yeast, there is evidence for a second, error-free, pathway that requires modification of PCNA with non-proteolytic lysine 63-linked polyubiquitin (K63-polyUb) chains. Here we demonstrate that formation of K63-polyUb chains protects human cells against translesion synthesis–induced mutations by promoting recovery of blocked replication forks through an alternative error-free mechanism. Furthermore, we show that polyubiquitination of PCNA occurs in UV-irradiated human cells. Our findings indicate that K63-polyubiquitination guards against environmental carcinogenesis and contributes to genomic stability.
Genome instability is associated with increased cancer risk, and thus considerable effort has been put into unraveling the mechanisms underlying genome surveillance. Guarding the integrity of DNA are a number of DNA-repair and cell cycle–control systems. Insight into how these pathways become activated is crucially important to the understanding of carcinogenesis and in the development of cancer treatments. This study concerns a distinct pathway that promotes the tolerance of DNA damage during its replication phase. Prior attempts to investigate this pathway in human cells have been difficult due to extensive redundancy in the genes that carry out this process. Previous knowledge from lower organisms suggested the requirement for enzymes capable of constructing a chain of ubiquitin molecules linked in a specific manner. The authors used a novel approach to disrupt the formation of these ubiquitin chains in human cells and found that this caused a significant increase in mutations after exposure to UV light. Several lines of evidence implicate a family of error-prone enzymes, called translesion synthesis polymerases, in the formation of these mutations. Furthermore, they provide evidence suggesting that proliferating cell nuclear antigen (PCNA), a protein found at sites of replication, is the relevant target of these chains in human cells. These findings indicate that polyubiquitination guards against environmental carcinogenesis and contributes to genomic stability.
Ionizing radiation exposure induces highly lethal DNA double-strand breaks (DSBs) in all phases of the cell cycle. After DSBs are detected by the cellular machinery, these breaks are repaired by either of two mechanisms: (1) nonhomologous end joining (NHEJ), which re-ligates the broken ends of the DNA and (2) homologous recombination (HR), that makes use of an undamaged identical DNA sequence as a template to maintain the fidelity of DNA repair. DNA DSB repair must occur within the context of the natural cellular DNA structure. Among the major factors influencing DNA organization are specific histone and nonhistone proteins that form chromatin. The overall chromatin structure regulates DNA damage responses since chromatin status can impede DNA damage site access by repair proteins. During the process of DNA DSB repair, several chromatin alterations are required to sense damage and facilitate accessibility of the repair machinery. The DNA DSB response is also facilitated by hierarchical signaling networks that orchestrate chromatin structural changes that may coordinate cell-cycle checkpoints involving multiple enzymatic activities to repair broken DNA ends. During DNA damage sensing and repair, histones undergo posttranslational modifications (PTMs) including phosphorylation, acetylation, methylation and ubiquitylation. Such histone modifications represent a histone code that directs the recruitment of proteins involved in DNA damage sensing and repair processes. In this review, we summarize histone modifications that occur during DNA DSB repair processes.
DNA is continuously exposed to many different damaging agents such as environmental chemicals, UV light, ionizing radiation, and reactive cellular metabolites. DNA lesions can result in different phenotypical consequences ranging from a number of diseases, including cancer, to cellular malfunction, cell death, or aging. To counteract the deleterious effects of DNA damage, cells have developed various repair systems, including biochemical pathways responsible for the removal of single-strand lesions such as base excision repair (BER) and nucleotide excision repair (NER) or specialized polymerases temporarily taking over lesion-arrested DNA polymerases during the S phase in translesion synthesis (TLS). There are also other mechanisms of DNA repair such as homologous recombination repair (HRR), nonhomologous end-joining repair (NHEJ), or DNA damage response system (DDR). This paper reviews bioinformatics resources specialized in disseminating information about DNA repair pathways, proteins involved in repair mechanisms, damaging agents, and DNA lesions.
BRCA1 promotes DNA repair through interactions with multiple proteins, including CtIP and FANCJ (also known as BRIP1/BACH1). While CtIP facilitates DNA end resection when de-acetylated, the function of FANCJ in repair processing is less well defined. Here, we report that FANCJ is also acetylated. Preventing FANCJ acetylation at lysine 1249 does not interfere with the ability of cells to survive DNA interstrand crosslinks (ICLs). However, resistance is achieved with reduced reliance on recombination. Mechanistically, FANCJ acetylation facilitates DNA end processing required for repair and checkpoint signaling. This conclusion was based on the finding that FANCJ and its acetylation were required for robust RPA foci formation, RPA phosphorylation, and Rad51 foci formation in response to camptothecin (CPT). Furthermore, both preventing and mimicking FANCJ acetylation at lysine 1249 disrupts FANCJ function in checkpoint maintenance. Thus, we propose that the dynamic regulation of FANCJ acetylation is critical for robust DNA damage response, recombination-based processing, and ultimately checkpoint maintenance.
The BRCA1–Fanconi anemia (FA) pathway is required for both tumor suppression and cell survival, particularly following treatment with DNA damaging agents that induce DNA interstrand crosslinks (ICLs). ICL processing by the BRCA–FA pathway includes promotion of homologous recombination (HR) and DNA damage tolerance through translesion synthesis. However, little is known about how the BRCA–FA pathway or these ICL processing mechanisms are regulated. Here, we identify acetylation as a DNA damage–dependent regulator of the BRCA–FA protein, FANCJ. FANCJ acetylation at lysine 1249 is enhanced by expression of the histone acetyltransferase CBP and reduced by expression of histone deacetylases HDAC3 or SIRT1. Furthermore, acetylation on endogenous FANCJ is induced upon treatment of cells with agents that generate DNA lesions. Consistent with this post-translation event regulating FANCJ function during cellular DNA repair, preventing FANCJ acetylation skews ICL processing. Cells have reduced reliance on HR factor Rad54 and greater reliance on translesion synthesis polymerase polη. Our data indicate that FANCJ acetylation contributes to DNA end processing that is required for HR. Furthermore, resection-dependent checkpoint maintenance relies on the dynamic regulation of FANCJ acetylation. The implication of these findings is that FANCJ acetylation contributes to DNA repair choice within the BRCA–FA pathway.
The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria.
In this study, we employed a bioinformatic approach, to analyse and describe the open reading frames potentially related to DNA repair from the genome of the alpha-proteobacterium Caulobacter crescentus. This was performed by comparison with known DNA repair related genes found in public databases. As expected, although C. crescentus and E. coli bacteria belong to separate phylogenetic groups, many of their DNA repair genes are very similar. However, some important DNA repair genes are absent in the C. crescentus genome and other interesting functionally related gene duplications are present, which do not occur in E. coli. These include DNA ligases, exonuclease III (xthA), endonuclease III (nth), O6-methylguanine-DNA methyltransferase (ada gene), photolyase-like genes, and uracil-DNA-glycosylases. On the other hand, the genes imuA and imuB, which are involved in DNA damage induced mutagenesis, have recently been described in C. crescentus, but are absent in E. coli. Particularly interesting are the potential atypical phylogeny of one of the photolyase genes in alpha-proteobacteria, indicating an origin by horizontal transfer, and the duplication of the Ada orthologs, which have diverse structural configurations, including one that is still unique for C. crescentus.
The absence and the presence of certain genes are discussed and predictions are made considering the particular aspects of the C. crescentus among other known DNA repair pathways. The observed differences enlarge what is known for DNA repair in the Bacterial world, and provide a useful framework for further experimental studies in this organism.
DNA repair and other chromatin-associated processes are carried out by enzymatic macromolecular complexes that assemble at specific sites on the chromatin fiber. How the rate of these molecular machineries is regulated by their constituent parts is poorly understood. Here we quantify nucleotide-excision DNA repair in mammalian cells and find that, despite the pathways' molecular complexity, repair effectively obeys slow first-order kinetics. Theoretical analysis and data-based modeling indicate that these kinetics are not due to a singular rate-limiting step. Rather, first-order kinetics emerge from the interplay of rapidly and reversibly assembling repair proteins, stochastically distributing DNA lesion repair over a broad time period. Based on this mechanism, the model predicts that the repair proteins collectively control the repair rate. Exploiting natural cell-to-cell variability, we corroborate this prediction for the lesion-recognition factor XPC and the downstream factor XPA. Our findings provide a rationale for the emergence of slow time scales in chromatin-associated processes from fast molecular steps and suggest that collective rate control might be a widespread mode of robust regulation in DNA repair and transcription.
The nucleotide-excision repair pathway removes mutagen-inflicted DNA lesions from the genome. Repair proteins recognize DNA lesions and form multi-protein complexes that catalyze the excision of the lesion and the re-synthesis of the excised part. Imaging the dynamics of fluorescently labeled repair proteins in living human cells has revealed that all factors continuously and rapidly exchange at repair sites. We asked how this dynamic mode of protein-complex assembly shapes the repair process. Measuring repair DNA synthesis in intact cells, we obtained a surprisingly simple result. Over the entire process, the rate is proportional to the amount of DNA lesions, where the proportionality factor is a single ‘slow’ rate constant. Such kinetic behavior is often regarded as evidence for a rate-limiting step, but we show here that it is an emergent property of the dynamic interplay of many repair proteins. As a consequence, the rate of DNA repair is a systems property that is controlled collectively by the expression levels of all repair factors. Given that transcription in living cells has similar dynamic features – rapidly exchanging components of the transcription machinery and slow bursts of mRNA synthesis – collective rate control might be a general property of chromatin-associated molecular machines.
Global genome nucleotide excision repair removes DNA damage from transcriptionally silent regions of the genome. Relatively little is known about the molecular events that initiate and regulate this process in the context of chromatin. We've shown that, in response to UV radiation–induced DNA damage, increased histone H3 acetylation at lysine 9 and 14 correlates with changes in chromatin structure, and these alterations are associated with efficient global genome nucleotide excision repair in yeast. These changes depend on the presence of the Rad16 protein. Remarkably, constitutive hyperacetylation of histone H3 can suppress the requirement for Rad7 and Rad16, two components of a global genome repair complex, during repair. This reveals the connection between histone H3 acetylation and DNA repair. Here, we investigate how chromatin structure is modified following UV irradiation to facilitate DNA repair in yeast. Using a combination of chromatin immunoprecipitation to measure histone acetylation levels, histone acetylase occupancy in chromatin, MNase digestion, or restriction enzyme endonuclease accessibility assays to analyse chromatin structure, and finally nucleotide excision repair assays to examine DNA repair, we demonstrate that global genome nucleotide excision repair drives UV-induced chromatin remodelling by controlling histone H3 acetylation levels in chromatin. The concerted action of the ATPase and C3HC4 RING domains of Rad16 combine to regulate the occupancy of the histone acetyl transferase Gcn5 on chromatin in response to UV damage. We conclude that the global genome repair complex in yeast regulates UV-induced histone H3 acetylation by controlling the accessibility of the histone acetyl transferase Gcn5 in chromatin. The resultant changes in histone H3 acetylation promote chromatin remodelling necessary for efficient repair of DNA damage. Recent evidence suggests that GCN5 plays a role in NER in human cells. Our work provides important insight into how GG-NER operates in chromatin.
Protection against genotoxic insult requires a network of DNA damage responses, including DNA repair. Inherited DNA repair defects cause severe clinical consequences including extreme cancer susceptibility. Despite detailed mechanistic understanding of the core reactions, little is known about the molecular events that initiate and regulate these processes as they occur in chromatin. We study the conserved nucleotide excision repair pathway in Saccharomyces cerevisiae. This pathway removes a broad spectrum of DNA damages including UV radiation–induced damage. Patients with mutations in genes involved in this process suffer dramatically elevated levels of skin and other cancers. Here we demonstrate how a group of genes involved in repair of transcriptionally silent regions of the genome, a process called global genome repair, modifies chromatin structure following UV irradiation to promote efficient removal of DNA damage from the genome. We show that the concerted action of global genome repair genes combine to regulate histone acetyl transferase accessibility to the chromatin in response to UV damage. In this way, global genome repair regulates histone H3 acetylation status, which ultimately regulates chromatin structure promoting efficient DNA repair in the genome. Our results contribute a significant advance in our understanding of how chromatin is processed during DNA repair.
The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5′-to-3′ resection of DNA ends, generating 3′ single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair.
A double-strand break (DSB) is a devastating form of DNA damage. Fortunately, cells are equipped with two DSB repair pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ). The Mre11-Rad50-Nbs1 (MRN) protein complex recognizes DSBs and initiates HR repair. The Mre11 subunit harbors a nuclease domain that is essential for repair in fission yeast and mammals, although the function is unknown. Here we show that Mre11 nuclease activity is required to release the Ku complex from DNA ends in fission yeast. While the initiation of repair, i.e. the generation of single-stranded DNA (ssDNA) overhangs in Mre11-nuclease dead mutants, is unaffected, we find that an essential downstream step involving the localization of Replication Protein A (RPA) to ssDNA is substantially decreased due to the inability to release Ku and MRN from the DNA end. In contrast, a DNA processing factor called Ctp1, which binds to Nbs1, is essential for the initiation of repair as well as the release of Ku and MRN from DNA ends. Importantly, we find that efficient localization of RPA, which is essential for efficient DSB repair by HR, requires the release of Ku and MRN from the DNA by the combined action of Mre11 nuclease and Ctp1.
Germline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs) can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR) and DSB repair (DSBR) within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to γ-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline.
Proper response to DNA damage and repair of DNA double-strand breaks (DSBs) is important to maintain genomic integrity and promote both accurate chromosome segregation and tumor suppression. Here we define the roles of a previously uncharacterized and conserved protein, ZTF-8, which is required for proper DNA damage checkpoint activation as well as DSB repair. Specifically, we provide a direct demonstration that ZTF-8 participates in both mitotic and meiotic DSB repair and in the meiotic DNA damage checkpoint via interacting with the 9-1-1 complex in the C. elegans germline. We propose that ZTF-8 is involved in promoting repair at blocked replication fork sites and meiotic DSBs in part by transducing DNA damage checkpoint signaling via the 9-1-1 DNA damage response complex.
To determine the level of expression for mRNAs that regulate DNA repair activity in oocytes at the germinal vesicle (GV) stage. Reactive oxygen species (ROS) have been shown to play a major role in the appearance of deleterious DNA decays, and this study focuses on the repair of damage linked to decay caused by the action of ROS. The oocyte needs a mechanism for repairing DNA decays in the early preimplantation embryo before the onset of genomic activation, since in the absence of repair, residual DNA damage would lead to either apoptosis or tolerance. Tolerance of DNA damage is a source of potential mutations.
GV oocytes were selected for this study, both for the ethical reason that they are unsuitable for patient treatment, and because no transcription takes place during the period from GV to MII and then prior to genomic activation. The GV oocyte is therefore a good model for looking at DNA during the first cleavages of early preimplantation development. Six cohorts of GV oocytes were pooled for extraction of mRNA; the DNA was analysed using Affimetrix HG-UG133 Plus 2, containing 54,675 probe sets; spike and housekeeping genes were also added as internal controls.
In GV oocytes, DNA repair pathways for oxidized bases are redundant. One step repair procedure (OSR), BER (base excision repair), MMR (mismatch repair) and NER (Nucleotide excision repair) are present. All the recognition proteins are also present. The chromatin assembly factors necessary for the maintenance of genomic stability are highly expressed.
Gene expression analysis shows that the oocyte does not allow a high level of tolerance for DNA decays. This regulatory mechanism should avoid transmitting mutations into the next generation.
Human oocyte; ROS; mRNA; DNA repair
DNA interstrand crosslinks (ICLs) are among the most cytotoxic types of DNA damage, and thus ICL-inducing agents such as cyclophosphamide, melphalan, cisplatin, psoralen and mitomycin C have been used clinically as anti-cancer drugs for decades. ICLs can also be formed endogenously as a consequence of cellular metabolic processes. ICL-inducing agents continue to be among the most effective chemotherapeutic treatments for many cancers; however, treatment with these agents can lead to secondary malignancies, in part due to mutagenic processing of the DNA lesions. The mechanisms of ICL repair have been characterized more thoroughly in bacteria and yeast than in mammalian cells. Thus, a better understanding the molecular mechanisms of ICL processing offers the potential to improve the efficacy of these drugs in cancer therapy. In mammalian cells it is thought that ICLs are repaired by the coordination of proteins from several pathways, including nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), homologous recombination (HR), translesion synthesis (TLS), and proteins involved in Fanconi anemia (FA). In this review, we focus on the potential functions of NER, MMR, and HR proteins in the repair of and response to ICLs in human cells and in mice. We will also discuss a unique approach, using psoralen covalently linked to triplex-forming oligonucleotides to direct ICLs to specific sites in the mammalian genome.
Psoralen; DNA interstrand crosslink; triplex; DNA repair
DNA repair is essential for routine monitoring and repair of damage imparted to our genetic material by exposure to endogenous and exogenous carcinogens, including reactive oxygen species, UV light, and chemicals such as those found in cigarette smoke. Without DNA repair pathways, the continual assault on our DNA would be highly mutagenic and the risk of cancer increased. Paradoxically, the same pathways that help prevent cancer development are detrimental to the efficacy of DNA-damaging cancer therapeutics such as cisplatin. Recent studies demonstrate the inverse relationship between DNA repair capacity and efficacy of platinum-based chemotherapeutics: increased DNA repair capacity leads to resistance, while decreased capacity leads to increased sensitivities. Cisplatin's cytotoxic effects are mediated by formation of intrastrand DNA crosslinks, which are predominantly repaired via the nucleotide excision repair (NER) pathway. In an effort to personalize the treatment of cancers based on DNA repair capacity, we developed an ELISA-based assay to measure NER activity accurately and reproducibly as a prognostic for platinum-based treatments. Here we present an overview of DNA repair and its link to cancer and therapeutics. We also present data demonstrating the ability to detect the proteins of the pre-incision complex within the NER pathway from cell and tissue extracts. Antioxid. Redox Signal. 14, 2465–2477.
In a living cell, a multiply damaged site in DNA is thought to be processed by several different pathways simultaneously or sequentially. Under this situation, the cellular response to the lesion cluster might depend on the order of repair processes because the configuration of the lesions will be modified by the reaction of initial repair protein, affecting the DNA-binding or lesion-excision activities of latter repair proteins. For example, a cluster comprised an AP site or SSB and base lesions is formed after one of the lesions in a base lesion cluster is excised by a glycosylase. It has been predicted that the enzymatic activity of a protein binding to a lesion could be affected by a second lesion located nearby. In the present study, we investigate whether initial base excision repair process affects secondary process on the clustered DNA damage site produced by high linear energy transfer irradiation.
Plasmid DNA (pUC18) used as model DNA were irradiated in a solution (10 mM Tris and 1 mM EDTA) with C6+ ions (60 keV/µm) obtained from HIMAC (NIRS, Chiba, Japan) according to our previous method [
1], or He2+ ions from TIARA (JAEA, Takasaki, Japan) in a fully hydrated film [
2]. We used two base excision repair enzymes to examine the order effect of BER processes, Nth and Fpg, which convert pyrimidine and purine lesions to an SSB, respectively. After exposure, the sample was incubated with Nth (or Fpg) at 37°C for 30 min. Then, we added another enzyme Fpg (or Nth) and incubate for another 30 min. We also simultaneously treated the DNA sample with both enzymes for 1 h. After the incubation, the samples were analysed by agarose (1%) gel electrophoresis to determine the yield of conformational changes of the plasmid from closed circular form to open or linear form. These changes show productions of an enzymatically induced SSB (the former) or DSB (the latter).
A typical example of the dependence of the loss of closed circular form DNA on radiation dose is shown in Fig. 1a for carbon ion irradiation. The reciprocal of the slope of each graph shows yield of prompt SSBs [n(SSB)prompt] for the sample without any enzymatic treatment, or [n(SSB)prompt] + enzymatically induced SSBs [n(SSB)Nth(or Fpg, or Nth+Fpg)]. The SSB yield for the sample treated with Nth first and then Fpg is very slightly less than that of other treatments (<10%). The dose responses of the linear form DNA production are shown in Fig. 1b. There were no significant differences among the enzymatic treatments. These results indicate that the configuration change of the cluster by the first enzymatic treatment does not significantly influence the activity of secondary enzyme. The most of base lesion clusters produced in the track of carbon ions might be converted to strand breaks, some of which form double-strand breaks (DSBs), by glycosylase activity in living cells.
Fig. 1.Dependence of (a) the loss of closed-circular DNA and (b) the number of linear form DNA DSB determined from the fraction of the linear form of DNA, on carbon ion dose. The arrows indicate the order of the enzymatic treatments.
clustered DNA damage; base excision repair; high LET radiation; carbon ion beam
The DNA helicase activity of the xeroderma pigmentosum D protein, a crucial subunit of TFIID, is only needed for its role in DNA repair, not for transcription.
The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER). Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue) to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription.
The multiprotein complex TFIIH is crucially involved in two fundamental cellular processes—the transcription of genes by RNA polymerase II and the repair of UV-induced DNA damage by a mechanism called nucleotide excision repair (NER). The xeroderma pigmentosum complementation group D (XPD) helicase, which is mutated in the eponymous human photosensitivity and cancer syndrome, is an important subunit of TFIIH, where it is assumed to act as a helicase, unwinding the DNA double helix. In our study, we show that XPD assumes an entirely different role in transcription and NER. In the case of repair, this protein works as an enzyme, requiring all its known functional properties. For transcription, however, none of the enzymatic functions is essential and XPD switches from an enzyme to a structural protein whose job is merely to preserve the integrity of the TFIIH complex. We have shown thus that the helicase activity of the XPD protein is exclusively devoted to repair processes and could be targeted by drugs without affecting transcription.
Deficiencies in DNA repair due to inherited germ-line mutations in DNA repair genes cause increased risk of gastrointestinal (GI) cancer. In sporadic GI cancers, mutations in DNA repair genes are relatively rare. However, epigenetic alterations that reduce expression of DNA repair genes are frequent in sporadic GI cancers. These epigenetic reductions are also found in field defects that give rise to cancers. Reduced DNA repair likely allows excessive DNA damages to accumulate in somatic cells. Then either inaccurate translesion synthesis past the un-repaired DNA damages or error-prone DNA repair can cause mutations. Erroneous DNA repair can also cause epigenetic alterations (i.e., epimutations, transmitted through multiple replication cycles). Some of these mutations and epimutations may cause progression to cancer. Thus, deficient or absent DNA repair is likely an important underlying cause of cancer. Whole genome sequencing of GI cancers show that between thousands to hundreds of thousands of mutations occur in these cancers. Epimutations that reduce DNA repair gene expression and occur early in progression to GI cancers are a likely source of this high genomic instability. Cancer cells deficient in DNA repair are more vulnerable than normal cells to inactivation by DNA damaging agents. Thus, some of the most clinically effective chemotherapeutic agents in cancer treatment are DNA damaging agents, and their effectiveness often depends on deficient DNA repair in cancer cells. Recently, at least 18 DNA repair proteins, each active in one of six DNA repair pathways, were found to be subject to epigenetic reduction of expression in GI cancers. Different DNA repair pathways repair different types of DNA damage. Evaluation of which DNA repair pathway(s) are deficient in particular types of GI cancer and/or particular patients may prove useful in guiding choice of therapeutic agents in cancer therapy.
Epigenetic; DNA damage; DNA repair; DNA repair deficiency disorders; Epimutation; Genomic instability; Germ-line mutation; MicroRNAs; Precancerous conditions; Gastrointestinal cancer
Efficient mechanisms for rejoining of DNA double-strand breaks (DSBs) are vital because misrepair of such lesions leads to mutation, aneuploidy and loss of cell viability. DSB repair is mediated by proteins acting in two major pathways, called homologous recombination and nonhomologous end-joining. Repair efficiency is also modulated by other processes such as sister chromatid cohesion, nucleosome remodeling and DNA damage checkpoints. The total number of genes influencing DSB repair efficiency is unknown.
To identify new yeast genes affecting DSB repair, genes linked to gamma radiation resistance in previous genome-wide surveys were tested for their impact on repair of site-specific DSBs generated by in vivo expression of EcoRI endonuclease. Eight members of the RAD52 group of DNA repair genes (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11 and XRS2) and 73 additional genes were found to be required for efficient repair of EcoRI-induced DSBs in screens utilizing both MATa and MATα deletion strain libraries. Most mutants were also sensitive to the clastogenic chemicals MMS and bleomycin. Several of the non-RAD52 group genes have previously been linked to DNA repair and over half of the genes affect nuclear processes. Many proteins encoded by the protective genes have previously been shown to associate physically with each other and with known DNA repair proteins in high-throughput proteomics studies. A majority of the proteins (64%) share sequence similarity with human proteins, suggesting that they serve similar functions.
We have used a genetic screening approach to detect new genes required for efficient repair of DSBs in Saccharomyces cerevisiae. The findings have spotlighted new genes that are critical for maintenance of genome integrity and are therefore of greatest concern for their potential impact when the corresponding gene orthologs and homologs are inactivated or polymorphic in human cells.
EcoRI; Homologous recombination; End-joining; Double-strand break; Bleomycin; MMS; Radiation; RAD52; Gene ontology (GO); Overlapping genes
Eukaryotic cells have developed mechanisms to prevent genomic instability, such as DNA damage detection and repair, control of cell cycle progression and cell death induction. The bifunctional compound furocumarin 8-methoxypsoralen (8-MOP) is widely used in the treatment of various inflammatory skin diseases. In this review, we summarize recent data about the role of chromatin remodeling in the repair of DNA damage induced by treatment with 8-methoxypsoralen plus UVA (8-MOP+UVA), focusing on repair proteins in budding yeast Saccharomyces cerevisiae, an established model system for studying DNA repair pathways. The interstrand crosslinks (ICL) formed by the 8-MOP+UVA treatment are detrimental lesions that can block transcription and replication, leading to cell death if not repaired. Current data show the involvement of different pathways in ICL processing, such as nucleotide excision repair (NER), base excision repair (BER), translesion repair (TLS) and double-strand break repair. 8-MOP+UVA treatment in yeast enhances the expression of genes involved in the DNA damage response, double strand break repair by homologous replication, as well as genes related to cell cycle regulation. Moreover, alterations in the expression of subtelomeric genes and genes related to chromatin remodeling are consistent with structural modifications of chromatin relevant to DNA repair. Taken together, these findings indicate a specific profile in 8-MOP+UVA responses related to chromatin remodeling and DNA repair.
DNA repair; psoralen; chromatin remodeling; histones; DNA interstrand cross-links