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.
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
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.
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 damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an α-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster–containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.
Preserving the structural integrity of DNA, and hence the genetic information stored in this molecule, is essential for cellular survival. It is estimated that the DNA in each human cell acquires about 104 lesions per day. Consequently, efficient DNA repair mechanisms have evolved to protect the genome. One of these DNA repair mechanisms, nucleotide excision repair (NER), is present in all organisms and is unique in its ability to repair a broad range of damage. In humans, NER is the major repair mechanism protecting DNA from damage induced by ultraviolet light. Defects in the genes and proteins responsible for NER can lead to one of three severe diseases: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. The XPD protein is one of the key components of a ten-protein complex and is essential to initiate NER. In particular, the XPD protein verifies the presence of damage to the DNA and thereby allows DNA repair to proceed. We have solved the 3-dimensional structure of the XPD protein, and show how XPD has assembled several domains to form a donut-shaped molecule, which is able to separate two DNA strands and scan the DNA for damage. The structure also helps to explain why some of the mutations that have been identified in humans are associated with disease.
The structure of the DNA repair protein XPD provides insights into how the protein binds and recognizes damaged DNA and how mutations inXPD disrupt its function and lead to disease.
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 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.
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 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.
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.
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.
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.
DNAtraffic (http://dnatraffic.ibb.waw.pl/) is dedicated to be a unique comprehensive and richly annotated database of genome dynamics during the cell life. It contains extensive data on the nomenclature, ontology, structure and function of proteins related to the DNA integrity mechanisms such as chromatin remodeling, histone modifications, DNA repair and damage response from eight organisms: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Escherichia coli and Arabidopsis thaliana. DNAtraffic contains comprehensive information on the diseases related to the assembled human proteins. DNAtraffic is richly annotated in the systemic information on the nomenclature, chemistry and structure of DNA damage and their sources, including environmental agents or commonly used drugs targeting nucleic acids and/or proteins involved in the maintenance of genome stability. One of the DNAtraffic database aim is to create the first platform of the combinatorial complexity of DNA network analysis. Database includes illustrations of pathways, damage, proteins and drugs. Since DNAtraffic is designed to cover a broad spectrum of scientific disciplines, it has to be extensively linked to numerous external data sources. Our database represents the result of the manual annotation work aimed at making the DNAtraffic much more useful for a wide range of systems biology applications.
The nucleotide excision repair pathway catalyzes the removal of bulky adduct damage from DNA and requires the activity of more than 30 individual proteins and complexes. A diverse array of damage can be recognized and removed by the NER pathway including UV-induced adducts and intrastrand adducts induced by the chemotherapeutic compound cisplatin. The recognition of DNA damage is complex and involves a series of proteins including the xeroderma pigmentosum group A and C proteins and the UV-damage DNA binding protein. The xeroderma pigmentosum group A protein is unique in the sense that it is required for both transcription coupled and global genomic nucleotide excision repair. In addition, xeroderma pigmentosum group A protein is required for the removal of all types of DNA lesions repaired by nucleotide excision repair. Considering its importance in the damage recognition process, the minimal information available on the mechanism of DNA binding and the potential that inhibition of xeroderma pigmentosum group A protein could enhance the therapeutic efficacy of platinum based anticancer drugs, we sought to identify and characterize small molecule inhibitors of the DNA binding activity of the xeroderma pigmentosum group A protein. In silico screening of a virtual small molecule library resulted in the identification of a class of molecules confirmed to inhibit the xeroderma pigmentosum group A protein-DNA interaction. Biochemical analysis of inhibition with varying DNA substrates revealed a common mechanism of xeroderma pigmentosum group A protein DNA binding to single-stranded DNA and cisplatin-damaged DNA.
Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5′ to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-β). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-β, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-β based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-β located downstream of APEX1 (3′ to the damaged site) and three with pol-β located upstream of APEX1 (5′ to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (∼−10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-β at the 3′-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-β in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-β in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3′ side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available.
Oxidative damage to DNA happens in every cell as a consequence of the life process. Such damage can inhibit DNA replication and RNA transcription; if not repaired, it can lead to cancer. Consequently, all cells contain an important mechanism for identification and repair of oxidative lesions. Two proteins figure prominently: AP endonuclease 1, which cleaves the damaged site, and DNA polymerase beta, which inserts a new nucleotide to replace the damaged one. While several biochemical studies indicate interaction between the two proteins, the details of the interaction remain unknown. Here, we develop and apply a new methodology to predict the most likely protein-protein interface between the two proteins. The methodology relies on the assumption, which is validated by experimental evidence, that both proteins must bind to DNA in order to interact. Analysis of the simulated protein behavior in water allowed us to suggest how protein interaction might be coupled to conformational changes in DNA polymerase beta. Further comparative analysis identified coordinated mutations of specific residues at the predicted interfaces. This method can be applied to predict interaction details for any protein pair as long as the proteins in the pair are associated with DNA during the interaction.
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.
Polyploidy is frequent in nature and is a hallmark of cancer cells, but little is known about the strategy of DNA repair in polyploid organisms. We have studied DNA repair in the polyploid archaeon Haloferax volcanii, which contains up to 20 genome copies. We have focused on the role of Mre11 and Rad50 proteins, which are found in all domains of life and which form a complex that binds to and coordinates the repair of DNA double-strand breaks (DSBs). Surprisingly, mre11 rad50 mutants are more resistant to DNA damage than the wild-type. However, wild-type cells recover faster from DNA damage, and pulsed-field gel electrophoresis shows that DNA double-strand breaks are repaired more slowly in mre11 rad50 mutants. Using a plasmid repair assay, we show that wild-type and mre11 rad50 cells use different strategies of DSB repair. In the wild-type, Mre11-Rad50 appears to prevent the repair of DSBs by homologous recombination (HR), allowing microhomology-mediated end-joining to act as the primary repair pathway. However, genetic analysis of recombination-defective radA mutants suggests that DNA repair in wild-type cells ultimately requires HR, therefore Mre11-Rad50 merely delays this mode of repair. In polyploid organisms, DSB repair by HR is potentially hazardous, since each DNA end will have multiple partners. We show that in the polyploid archaeon H. volcanii the repair of DSBs by HR is restrained by Mre11-Rad50. The unrestrained use of HR in mre11 rad50 mutants enhances cell survival but leads to slow recovery from DNA damage, presumably due to difficulties in the resolution of DNA repair intermediates. Our results suggest that recombination might be similarly repressed in other polyploid organisms and at repetitive sequences in haploid and diploid species.
Most organisms contain only one or two copies of their genome, but in some species multiple copies are found. The presence of multiple genome copies (polyploidy) has profound implications for DNA repair and is frequently seen in cancer cells. We have studied DNA repair in the archaeon Haloferax volcanii, which contains up to 20 genome copies. Archaea are a third form of life distinct from bacteria and eukaryotes. We have focused on the DNA repair proteins Mre11 and Rad50, which are found in virtually all organisms and which in humans act to prevent cancer. Surprisingly, we have found that H. volcanii cells deficient in Mre11-Rad50 are more resistant to DNA damage than wild-type cells. The DNA damage resistance of mre11 rad50 mutant cells appears to be due to the exclusive use of homologous recombination, a DNA repair mechanism that is accurate but has the potential to generate genome rearrangements that require time to resolve. Correspondingly, we have found repair of DNA damage in mre11 rad50 mutants takes longer than in wild-type cells. Our results suggest that polyploid organisms employ a program of DNA repair that minimizes their reliance on homologous recombination.
Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5′-3′ exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA–related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.
Individuals with Fanconi anemia (FA) suffer from bone marrow failure and from elevated rates of haematological and solid malignancy. Moreover, FA patients exhibit extreme sensitivity to DNA interstrand cross-links (ICLs), but not other forms of DNA damage. Despite recent progress in identifying and characterising FA factors, little is known about the mechanistic basis of the ICL repair defect in FA. The identification and characterisation of FA–like pathways in simple model eukaryotes, amenable to genetic dissection, would clearly accelerate progress. Here, we have identified an ICL repair pathway in budding yeast that has significant similarities to the FA pathway and that acts in parallel to an established pathway controlled by the Pso2 exonuclease. We have discovered that a key component of this pathway, the FANCM-like helicase, Mph1, interacts and collaborates with a mismatch repair factor (MutSα) and a novel nuclear DNA repair factor Mgm101 to control ICL repair. We also found that a central role of these factors is to recruit Exonuclease 1 (Exo1) to ICL-damaged chromatin, and propose that this factor acts redundantly with Pso2 to execute the exonucleolytic processing of ICLs. Our findings reveal new mechanistic insights into the control of ICL repair by FA–like proteins in an important model organism.
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.
This study reveals the molecular mechanism by which the nucleotide excision repair protein DDB2 prioritises excision of UV-induced DNA lesions in the nucleosome landscape.
How tightly packed chromatin is thoroughly inspected for DNA damage is one of the fundamental unanswered questions in biology. In particular, the effective excision of carcinogenic lesions caused by the ultraviolet (UV) radiation of sunlight depends on UV-damaged DNA-binding protein (UV-DDB), but the mechanism by which this DDB1-DDB2 heterodimer stimulates DNA repair remained enigmatic. We hypothesized that a distinctive function of this unique sensor is to coordinate damage recognition in the nucleosome repeat landscape of chromatin. Therefore, the nucleosomes of human cells have been dissected by micrococcal nuclease, thus revealing, to our knowledge for the first time, that UV-DDB associates preferentially with lesions in hypersensitive, hence, highly accessible internucleosomal sites joining the core particles. Surprisingly, the accompanying CUL4A ubiquitin ligase activity is necessary to retain the xeroderma pigmentosum group C (XPC) partner at such internucleosomal repair hotspots that undergo very fast excision kinetics. This CUL4A complex thereby counteracts an unexpected affinity of XPC for core particles that are less permissive than hypersensitive sites to downstream repair subunits. That UV-DDB also adopts a ubiquitin-independent function is evidenced by domain mapping and in situ protein dynamics studies, revealing direct but transient interactions that promote a thermodynamically unfavorable β-hairpin insertion of XPC into substrate DNA. We conclude that the evolutionary advent of UV-DDB correlates with the need for a spatiotemporal organizer of XPC positioning in higher eukaryotic chromatin.
Like all molecules in living organisms, DNA undergoes spontaneous decay and is constantly under attack by endogenous and environmental agents. Unlike other molecules, however, DNA—the blueprint of heredity—cannot be re-created de novo; it can only be copied. The original blueprint must therefore remain pristine. All kinds of DNA damage pose a health hazard. DNA lesions induced by the ultraviolet (UV) component of sunlight, for example, can lead to skin aging and skin cancer. A repair process known as nucleotide excision repair (NER) is dedicated to correcting this UV damage. Although the enzymatic steps of this repair process are known in detail, we still do not understand how it copes with the native situation in the cell, where the DNA is tightly wrapped around protein spools called nucleosomes. Our study has revealed the molecular mechanism by which an enigmatic component of NER called UV-DDB stimulates excision of UV-induced lesions in the landscape of nucleosome-packaged DNA in human skin cells. In particular, we describe how this accessory protein prioritizes, in space and time, which UV lesions in packaged DNA to target for repair by NER complexes, thus optimizing the repair process.
Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.
A deletion of the sbcC gene rendered Bacillus subtilis cells sensitive to DNA damage caused by Mitomycin C (MMC) or by gamma irradiation. The deletion of the sbcC gene in a recN mutant background increased the sensitivity of the single recN mutant strain. SbcC was also non-epistatic with AddAB (analog of Escherichia coli RecBCD), but epistatic with RecA. A deletion of the ykoV gene encoding the B. subtilis Ku protein in a sbcC mutant strain did not resulted in an increase in sensitivity towards MMC and gamma irradiation, but exacerbated the phenotype of a recN or a recA mutant strain. In exponentially growing cells, SbcC-GFP was present throughout the cells, or as a central focus in rare cases. Upon induction of DNA damage, SbcC formed 1, rarely 2, foci on the nucleoids. Different to RecN protein, which forms repair centers at any location on the nucleoids, SbcC foci mostly co-localized with the DNA polymerase complex. In contrast to this, AddA-GFP or AddB-GFP did not form detectable foci upon addition of MMC.
Our experiments show that SbcC plays an important role in the repair of DNA inter-strand cross-links (induced by MMC), most likely through HR, and suggest that NHEJ via Ku serves as a backup DNA repair system. The cell biological experiments show that SbcC functions in close proximity to the replication machinery, suggesting that SbcC may act on stalled or collapsed replication forks. Our results show that different patterns of localization exist for DNA repair proteins, and that the B. subtilis SMC proteins RecN and SbcC play distinct roles in the repair of DNA damage.
Previously, we have demonstrated that human oxidative DNA glycosylase NEIL1
excises photoactivated psoralen-induced monoadducts but not genuine
interstrand cross-links (ICLs) in duplex DNA. It has been postulated that the
repair of ICLs in mammalian cells is mainly linked to DNA replication and
proceeds via dual incisions in one DNA strand that bracket the cross-linked
site. This process, known as “unhooking,” enables strand
separation and translesion DNA synthesis through the gap, yielding a
three-stranded DNA repair intermediate composed of a short unhooked oligomer
covalently bound to the duplex. At present, the detailed molecular mechanism
of ICL repair in mammalian cells remains unclear. Here, we constructed and
characterized three-stranded DNA structures containing a single ICL as
substrates for the base excision repair proteins. We show that NEIL1 excises
with high efficiency the unhooked ICL fragment within a three-stranded DNA
structure. Complete reconstitution of the repair of unhooked ICL shows that it
can be processed in a short patch base excision repair pathway. The new
substrate specificity of NEIL1 points to a preferential involvement in the
replication-associated repair of ICLs. Based on these data, we propose a model
for the mechanism of ICL repair in mammalian cells that implicates the DNA
glycosylase activity of NEIL1 downstream of Xeroderma Pigmentosum group
F/Excision Repair Cross-Complementing 1 endonuclease complex (XPF/ERCC1) and
translesion DNA synthesis repair steps. Finally, our data demonstrate that
Nei-like proteins from Escherichia coli to human cells can excise
bulky unhooked psoralen-induced ICLs via hydrolysis of glycosidic bond between
cross-linked base and deoxyribose sugar, thus providing an alternative
heuristic solution for the removal of complex DNA lesions.
DNA repair is critical to resolve extrinsic or intrinsic DNA damage to ensure regulated gene transcription and DNA replication. These pathways control repair of double strand breaks, interstrand crosslinks, and nucleotide lesions occurring on single strands. Distinct DNA repair pathways are highly inter-linked for the fast and optimal DNA repair. A deregulation of DNA repair pathways may maintain and promote genetic instability and drug resistance to genotoxic agents in tumor cells by specific mechanisms that tolerate or rapidly bypass lesions to drive proliferation and abrogate cell death. Multiple Myeloma (MM) is a plasma cell disorder characterized by genetic instability and poor outcome for some patients, in which the compendium of DNA repair pathways has as yet not been assessed for a disease-specific prognostic relevance. We design a DNA repair risk score based on the expression of genes coding for proteins involved in DNA repair in MM cells. From a consensus list of 84 DNA repair genes, 17 had a bad prognostic value and 5 a good prognostic value for both event-free and overall survival of previously-untreated MM patients. The prognostic information provided by these 22 prognostic genes was summed within a global DNA repair score (DRScore) to take into account the tight linkage of repair pathways. DRscore was strongly predictive for both patients' event free and overall survivals. Also, DRscore has the potential to identify MM patients whose tumor cells are dependent on specific DNA repair pathways to design treatments that induce synthetic lethality by exploiting addiction to deregulated DNA repair pathways.
DNA repair; Multiple Myeloma; prognosis
Sequenced archaeal genomes contain a variety of bacterial and eukaryotic DNA repair gene homologs, but relatively little is known about how these microorganisms actually perform DNA repair. At least some archaea, including the extreme halophile Halobacterium sp. NRC-1, are able to repair ultraviolet light (UV) induced DNA damage in the absence of light-dependent photoreactivation but this 'dark' repair capacity remains largely uncharacterized. Halobacterium sp. NRC-1 possesses homologs of the bacterial uvrA, uvrB, and uvrC nucleotide excision repair genes as well as several eukaryotic repair genes and it has been thought that multiple DNA repair pathways may account for the high UV resistance and dark repair capacity of this model halophilic archaeon. We have carried out a functional analysis, measuring repair capability in uvrA, uvrB and uvrC deletion mutants.
Deletion mutants lacking functional uvrA, uvrB or uvrC genes, including a uvrA uvrC double mutant, are hypersensitive to UV and are unable to remove cyclobutane pyrimidine dimers or 6–4 photoproducts from their DNA after irradiation with 150 J/m2 of 254 nm UV-C. The UV sensitivity of the uvr mutants is greatly attenuated following incubation under visible light, emphasizing that photoreactivation is highly efficient in this organism. Phylogenetic analysis of the Halobacterium uvr genes indicates a complex ancestry.
Our results demonstrate that homologs of the bacterial nucleotide excision repair genes uvrA, uvrB, and uvrC are required for the removal of UV damage in the absence of photoreactivating light in Halobacterium sp. NRC-1. Deletion of these genes renders cells hypersensitive to UV and abolishes their ability to remove cyclobutane pyrimidine dimers and 6–4 photoproducts in the absence of photoreactivating light. In spite of this inability to repair UV damaged DNA, uvrA, uvrB and uvrC deletion mutants are substantially less UV sensitive than excision repair mutants of E. coli or yeast. This may be due to efficient damage tolerance mechanisms such as recombinational lesion bypass, bypass DNA polymerase(s) and the existence of multiple genomes in Halobacterium. Phylogenetic analysis provides no clear evidence for lateral transfer of these genes from bacteria to archaea.
Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB) repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ), single-strand annealing (SSA), and homology directed repair (HDR/GC). Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI–induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy.
Changes to the sequence of DNA, or mutations, can disrupt cellular growth control genes, which can lead to cancer development. Such mutations likely arise from damage to DNA that is repaired in a way that fails to restore the original sequence. One type of DNA damage is a chromosomal double-strand break. We have developed assays to measure how these breaks are repaired, and also how such repair can lead to mutations. In particular, we present an assay to measure a pathway of repair that results in deletion mutations, often with evidence of short homologous sequences at the repair junctions (alt-NHEJ). We have compared the genetic requirements of this repair pathway in relation to other pathways of repair that use extensive homology. We find that factors KU and CtIP appear to affect the initial stages of repair of each of these pathways, regardless of the length of homology. However, these pathways appear to diverge at later steps, as relates to the role of the repair factors RAD52, ERCC1, and RAD51. Given that mutations observed in some cancer cells are consistent with alt-NHEJ repair, these mechanistic descriptions provide models for how such mutations could arise in cancer.