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RAD51 supports spontaneous non-homologous recombination in mammalian cells, but not the corresponding process induced by topoisomerase inhibitors
Abstract
The RAD51 protein has been shown to participate in homologous recombination by promoting ATP-dependent homologous pairing
and strand transfer reactions. In the present study, we have investigated the possible involvement of RAD51 in non-homologous
recombination. We demonstrate that overexpression of CgRAD51 enhances the frequency of spontaneous non-homologous recombination in the hprt gene of Chinese hamster cells. However, the rate of non-homologous recombination induced by the topoisomerase inhibitors
campothecin and etoposide was not altered by overexpression of RAD51. These results indicate that the RAD51 protein may perform a function in connection with spontaneous non-homologous recombination
that is not essential to or not rate-limiting for non-homologous recombination induced by camptothecin or etoposide. We discuss
the possibility that the role played by RAD51 in non-homologous recombination observed here may not be linked to non-homologous
end-joining.
... Rad51 is a RecA homologous recombinase in eukaryotes, which is essential for the homologous recombination (HR) process either in meiosis or for repairing DNA double-strand breaks (DSBs) (Masson and West 2001;Baumann and West 1998). With the other similar family members, Rad51 is involved in the search of the homologous intact duplex, DNA pairing and strand exchange (Shinohara and Ogawa 1995;Baumann et al. 1996;Arnaudeau et al. 2001). In normal cells, Rad51 interacting proteins have been suggested to tune the expression of Rad51 recombinase (Richardson 2005;Thacker 2005;Raderschall et al. 2002;Maacke et al. 2000;Xia et al. 1997;Richardson et al. 2004). ...
... Rad51 has been demonstrated to be elevated in a number of tumor cell lines (Xia et al. 1997;Arnaudeau et al. 1999). Whether, overexpression of RAD51 in cells stimulates HR (Arnaudeau et al. 1999;Maacke et al. 2000;Martin et al. 2007;Vispe et al. 1998) or reduces HR (Arnaudeau et al. 2001), it may potentially lead to chromosome rearrangements (Klein 2008;Lundin et al. 2003). However, RAD51 may not to be an oncogene because it may be an essential gene, redundant gene, and/or independent of the BRCA1/BRCA2 tumor suppressor pathway (Schmutte et al. 1999). ...
Breast cancer (BC) is the most common cancer and the second leading cause of death among women worldwide. Only 10% of BC cases have been related to genetic predisposition. Rad51, a homologous recombination (HR) protein plays an important role in HR in meiosis and repairing DNA double-strand breaks. Expression of RAD51 may be a predictive biomarker in certain types of cancers. The exact mechanisms involved in the regulation of RAD51 expression are not fully understood, but certain transcription factors have been suggested to be the tuning mechanism of its expression. In this study, we propose that polymorphisms in the 5'-UTR promoter region of the RAD51 gene are prognostic factors for BC development. Direct sequencing of 106 samples from sporadic BC patients and 54 samples from a control group was performed. FFPE samples were the choice of sample collection, which might be a limitation of our study. Homologous variant T172T alone was found to be significantly associated with BC risk (OR 3.717, 95% CI 2.283-6.052, p < 0.0001). On the other hand, heterozygous G135C did not show any significant relationship with risk of sporadic BC (OR 1.598, 95% CI 0.5638-4.528, p > 0.05). Moreover, both variants; homozygous T172T and heterozygous G135C together; showed a significant relationship with sporadic BC susceptibility.
... RAD51 is a conserved universal recombinase that forms helical filaments at ssDNA and promotes double-strand repair of broken DNA. On the one hand, it binds to SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein 1 (SMARCAL1) at stalled replication forks to repair replication forks and promote fork inversion (Baumann and West, 1998;Arnaudeau, 2001). On the other hand, RAD51 protects newly synthesized DNA from nuclease degradation and pro. ...
Breast cancer and gynecological tumors seriously endanger women’s physical and mental health, fertility, and quality of life. Due to standardized surgical treatment, chemotherapy, and radiotherapy, the prognosis and overall survival of cancer patients have improved compared to earlier, but the management of advanced disease still faces great challenges. Recently, poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) have been clinically approved for breast and gynecological cancer patients, significantly improving their quality of life, especially of patients with BRCA1/2 mutations. However, drug resistance faced by PARPi therapy has hindered its clinical promotion. Therefore, developing new drug strategies to resensitize cancers affecting women to PARPi therapy is the direction of our future research. Currently, the effects of PARPi in combination with other drugs to overcome drug resistance are being studied. In this article, we review the mechanisms of PARPi resistance and summarize the current combination of clinical trials that can improve its resistance, with a view to identify the best clinical treatment to save the lives of patients.
Abstract
Breast cancer is a heterogeneous disease at morphologic and molecular levels, which is considered the most commonly occurring cancer in women. RAD51, a DNA-repairing protein, involves homologous recombination and has a vital role in genome stability. Polymorphism of the RAD51 gene, and its overexpression, has been proposed to be associated with the development of breast cancer. Overexpression of RAD51 in many types of human cancer including metastatic breast cancer may signify its potential use as a biomarker. Considering the numerous reports on the role of the 5'-UTR-RAD51 polymorphism in breast cancer, this study aimed to investigate the utility of RAD51 gene expression and its variants G135C and G172T as a possible foretelling factor of breast cancer development. DNA sequencing and immunohistochemistry of RAD51 were conducted on 103 samples from patients diagnosed with sporadic breast cancer and 80 samples from a control group. The results demonstrated that the RAD51 variants, G135C and G172T, were significantly presented in the breast cancer tissue compared with the control group. RAD51 expression was mainly shown in the cytoplasm of malignant cells (56% of cases) and significantly correlated with p53 and G135C, C135C variants. Moreover, the occurrence of the G172T variant was significantly associated with the expression of estrogen receptor. Interestingly, 21/26 (81%) of the triple-negative breast cancer showed G135C and C135C genotypes that were significantly associated with the expression of RAD51 (73%). In conclusion, the G135C and C135C variants together with the cytoplasmic expression of RAD51 may have clinical potential as a prognostic predictor for breast cancer development and aggressiveness.
Introduction: We describe a new, third-generation of antibody-drug conjugates (ADCs) having a high drug payload against topoisomerase I, important for DNA function, and targeting selective tumor antigens, predominantly TROP-2.
Areas Covered: The historical development of ADCs is reviewed before presenting the current line of improved, third-generation ADCs targeting topoisomerase I, thus affecting DNA and causing double-stranded DNA breaks. Emphasis is given to explaining why sacituzumab govitecan represents a paradigm change in ADCs by achieving a high therapeutic index due to its novel target, TROP-2, an internalizing antigen/antibody, proprietary linker chemistry, and high drug payload, resulting in a high tumor concentration of the drug given in repeated doses with acceptable tolerability, particularly evidencing a lower percentage of “late” diarrhea than its prodrug, irinotecan. PubMed was used for the primary search conducted.
Expert Opinion: The properties and clinical results of third-generation ADCs, based on sacituzumab govitecan, are discussed, including prospects for future applications, particularly combination therapies with PARP inhibitors and immune checkpoint inhibitors. Since one topoisomerase I ADC has just received regulatory approval for HER-2⁺ breast cancer, and sacituzumab govitecan is under FDA review for accelerated approval in the therapy of triple-negative breast cancer, the prospects for these novel ADCs are discussed.
Natural and synthetic small molecules from the NCI Developmental Therapeutics Program (DTP) were employed in molecular dynamics-based docking with DNA repair proteins whose RNA-Seq based expression was associated with overall cancer survival (OS) after adjustment for the PCNA metagene. The compounds employed were required to elicit a sensitive response (vs. resistance) in more than half of the cell lines tested for each cancer. Methodological approaches included peptide sequence alignments and homology modeling for 3D protein structure determination, ligand preparation, docking, toxicity and ADME prediction. Docking was performed for unique lists of DNA repair proteins which predict OS for AML, cancers of the breast, lung, colon, and ovaries, GBM, melanoma, and renal papillary cancer. Results indicate hundreds of drug-like and lead-like ligands with best-pose binding energies less than −6 kcal/mol. Ligand solubility for the top 20 drug-like hits approached lower bounds, while lipophilicity was acceptable. Most ligands were also blood-brain barrier permeable with high intestinal absorption rates. While the majority of ligands lacked positive prediction for HERG channel blockage and Ames carcinogenicity, there was a considerable variation for predicted fathead minnow, honey bee, and Tetrahymena pyriformis toxicity. The computational results suggest the potential for new targets and mechanisms of repair inhibition and can be directly employed for in vitro and in vivo confirmatory laboratory experiments to identify new targets of therapy for cancer survival.
A series of critical pathways are responsible for the detection, signaling and restart of replication forks that encounter blocks during S-phase progression. Small base lesions may obstruct replication fork progression and processing, but the link between repair of small lesions and replication forks is unclear. In this study, we investigated an hypothesized role for DNA-PK, an important enzyme in DNA repair, in cellular responses to DNA replication stress. The enzyme catalytic subunit DNA-PKcs was phosphorylated on S2056 at sites of stalled replication forks in response to short hydroxyurea treatment. Using DNA fiber experiments, we found that catalytically active DNA-PK was required for efficient replication restart of stalled forks. Further, enzymatically active DNA-PK was also required for PARP-dependent recruitment of XRCC1 to stalled replication forks. This activity was enhanced by preventing Mre11-dependent DNA end resection, suggesting that XRCC1 must be recruited early to an unresected stalled fork. We also found that XRCC1 was required for effective restart of a subset of stalled replication forks. Overall, our work suggested that DNA-PK and PARP-dependent recruitment of XRCC1 is necessary to effectively protect, repair, and restart stalled replication forks, providing new insight into how genomic stability is preserved.
Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. It is well known that CML cells are genetically unstable. However, the mechanisms by which these cells acquire genetic alterations are poorly understood. Imatinib mesylate is the standard therapy for newly diagnosed CML patients. Imatinib mesylate targets the oncogenic kinase activity of BCR-ABL.
To study the gene expression profile of bone marrow hematopoietic cells in the same patients with CML before and 1 month after imatinib therapy.
Samples from patients with CML were analyzed using Affymetrix GeneChip Expression Arrays.
A total of 594 differentially expressed genes, most of which (393 genes) were downregulated, as a result of imatinib therapy were observed.
The blockade of oncoprotein Bcr-Abl by imatinib could cause a decrease in the expression of key DNA repair genes and substantially modify the expression profile of the bone marrow cells in the first days of therapy.
Rad51 plays a critical role in homologous recombination and correlates with many human malignancies. However, its role and clinical significance in esophageal squamous cell carcinoma (ESCC) has not been clarified. The purpose of this study was to explore the clinicopathological correlation and prognostic significance of Rad51 expression in a group of ESCC patients.
We evaluated Rad51 expression in 230 surgically resected ESCC specimens by immunochemistry using tissue microarray and correlated with clinicopathological features including post-operation survival.
There was no significant correlation between Rad51 expression and clinicopathological characteristics in terms of age, sex, tumor location, histologic grade, T, N categories, and TNM stage. The Kaplan-Meier survival analysis showed that high expression of Rad51 indicated a poorer disease free survival (DFS) of ESCC patients compared with the patients with low expression of Rad51 (P = 0.031), similar result also shown in overall survival (OS) analysis (P = 0.034). Furthermore, Rad51 expression could stratify node positive patients in DFS (P = 0.021) and OS (P = 0.035). Multivariate analysis confirmed the Rad51 expression was an independent prognostic factor for DFS (HR = 1.603, P = 0.013) and OS (HR = 1.555, P = 0.021) of ESCC patients.
Elevated expression of Rad51 is associated with poor prognosis for resectable ESCC patients.
The Werner syndrome protein (WRN) is a member of the human RecQ family DNA helicases implicated in the maintenance of genome stability. Loss of WRN gives rise to the Werner syndrome, a genetic disease characterised by premature aging and cancer predisposition. WRN plays a crucial role in the response to replication stress and significantly contributes to the recovery of stalled replication forks, although how this function is regulated is not fully appreciated. There is a growing body of evidence that WRN accomplishes its task in close connection with the replication checkpoint. In eukaryotic cells, the replication checkpoint response, which involves both the ATR and ATM kinase activities, is deputed to the maintenance of fork integrity and re-establishment of fork progression. Our recent findings indicate that ATR and ATM modulate WRN function at defined steps of the response to replication fork arrest. This review focuses on the novel evidence of a functional relationship between WRN and the replication checkpoint and how this cross-talk might contribute to prevent genome instability, a common feature of senescent and cancer cells.
RAD51 is a key enzyme of homologous recombination and repair of DNA double-strand breaks. RAD51 mRNA expression levels are significantly increased in laser-microdissected mammary simple carcinomas and their lymph node metastases when compared to adenomas or nonneoplastic mammary gland of the same dog. Here, RAD51 protein expression was analyzed by immunohistochemistry in paraffin-embedded mammary carcinomas and their lymph node metastases of 40 dogs, adenomas of 48 dogs, and nonneoplastic mammary gland of 88 dogs. Number of cells with nuclear RAD51 expression was significantly (P < or = .05) increased in carcinomas when compared to adenomas and metastases. In contrast, no significant differences in the number of RAD51-expressing cells were detected when metastases were compared with adenomas and nonneoplastic gland. RAD51 expression in carcinomas was correlated with expression in metastases but not with histologic grade. In conclusion, the increased number of RAD51-expressing cells in carcinomas might indicate genomic instability in these cells. Nevertheless, the increased RAD51 mRNA expression in metastases could not be confirmed by immunohistochemistry.
Targeted gene alteration (TGA) is a strategy for correcting single base mutations in the DNA of human cells that cause inherited disorders. TGA aims to reverse a phenotype by repairing the mutant base within the chromosome itself, avoiding the introduction of exogenous genes. The process of how to accurately repair a genetic mutation is elucidated through the use of single-stranded DNA oligonucleotides (ODNs) that can enter the cell and migrate to the nucleus. These specifically designed ODNs hybridize to the target sequence and act as a beacon for nucleotide exchange. The key to this reaction is the frequency with which the base is corrected; this will determine whether the approach becomes clinically relevant or not. Over the course of the last five years, workers have been uncovering the role played by the cells in regulating the gene repair process. In this essay, we discuss how the impact of the cell on TGA has evolved through the years and illustrate ways that inherent cellular pathways could be used to enhance TGA activity. We also describe the cost to cell metabolism and survival when certain processes are altered to achieve a higher frequency of repair.
Rad51 protein, involved in homologous recombination, is overexpressed in a variety of tumors, and its expression is correlated with a poor prognosis. Here we propose to exploit the overexpression of Rad51 in cancer cells to design a Rad51 promoter-based anticancer therapy. On average, Rad51 mRNA and protein levels are increased in cancer cells four- and sixfold, respectively. Serendipitously, we discovered that when the Rad51 ORF is replaced with another ORF, the difference in promoter activity between normal and cancer cells increases to an average of 840-fold with a maximum difference of 12,500-fold. This dramatic difference in activity has high therapeutic potential. We demonstrate that the fusion of Rad51 promoter to diphtheria toxin A (DTA) gene kills a variety of cancer cell types, including breast cancer, fibrosarcoma, and cervical cancer cells, with minimal effect on normal breast epithelial cells and normal fibroblasts. Our results suggest that therapies based on the Rad51 promoter will be highly tumor specific and open new avenues for targeting a broad range of cancers.
• cancer
• transcriptionally targeted therapy
Disruption of replication can lead to loss of genome integrity and increase of cancer susceptibility in mammals. Thus, a replication impediment constitutes a formidable challenge to these organisms. Recent studies indicate that homologous recombination (HR) plays an important role in suppressing genome instability and promoting cell survival after exposure to various replication inhibitors, including a topoisomerase I inhibitor, camptothecin (CPT). Here, we report that the deletion of RecQ helicase Recql5 in mouse ES cells and embryonic fibroblast (MEF) cells resulted in a significant increase in CPT sensitivity and a profound reduction in DNA replication after the treatment with CPT, but not other DNA-damaging agents. This CPT-induced cell death is replication dependent and occurs primarily after the cells had exited the first cell cycle after CPT treatment. Furthermore, we show that Recql5 functions nonredundantly with Rad51, a key factor for HR to protect mouse ES cells from CPT-induced cytotoxicity. These new findings strongly suggest that Recql5 plays an important role in maintaining active DNA replication to prevent the collapse of replication forks and the accumulation of DSBs in order to preserve genome integrity and to prevent cell death after replication stress as a result of topoisomerase I poisoning.
Camptothecins demonstrate a broad spectrum of antitumor activity. Although they are known to trap DNA topoisomerase I on DNA, form cleavable complexes, and generate DNA breaks upon collision with DNA or RNA polymerases, the precise mechanisms predictive for antitumor activity remain to be identified. Recent studies using panels of colorectal and breast cancer cell lines indicate that events downstream of cleavable complexes are more relevant. In this study, we chose SN-38, an active metabolite of irinotecan, to characterize DNA double strand breaks and repair mechanisms induced by this type of drugs using a human head and neck squamous cell carcinoma cell line A253. The results showed that 2-h exposure of cells to an IC(50) concentration of SN-38 induces biphasic DNA double-strand break (DSBs): an immediate phase, which was greatly reduced within 8 h, and a lagging phase, culminating 24 h after drug removal. Three DNA double-strand break repair protein complexes were activated: DNA-dependent protein kinase (DNA-PK), NBS1-MRE11-RAD50, and BRCA1. Aphidicolin, a DNA polymerase inhibitor, abolished both phase I DSBs and the activation of repair protein complexes, suggesting that they resulted from the collision between the cleavable complex and DNA polymerase of S-phase cells. This is in contrast to ionizing radiation-induced activation of DNA-PK and NBS1-MRE11-RAD50 complexes that occur predominantly among non-S-phase cells. The trigger for phase II DSBs cannot be abolished by aphidicolin. The data also indicate that DNA fragments in the size of 50 to 200 kilobases were detected in the lagging phase. This suggests that the late DNA DSBs were associated with apoptotic cell death.
Oncogenic tyrosine kinases (OTKs) are involved in the induction of many types of tumour, including haematological malignancies and cancers of the breast, prostate, colon and lung. Neoplastic cells that express OTKs are usually resistant to apoptosis that is induced by DNA-damaging agents, such as cytostatic drugs and irradiation, and they display genomic instability. So, what are the mechanisms involved, and what is the potential for overcoming OTK-mediated resistance in the clinic?
Homologous recombination (HR) and nonhomologous end joining (NHEJ) play overlapping roles in repair of DNA double-strand breaks
(DSBs) generated during the S phase of the cell cycle. Here, we characterized the involvement of HR and NHEJ in the rescue
of DNA replication forks arrested or slowed by treatment of hamster cells with hydroxyurea or thymidine. We show that the
arrest of replication with hydroxyurea generates DNA fragmentation as a consequence of the formation of DSBs at newly replicated
DNA. Both HR and NHEJ protected cells from the lethal effects of hydroxyurea, and this agent also increased the frequency
of recombination mediated by both homologous and nonhomologous exchanges. Thymidine induced a less stringent arrest of replication
and did not generate detectable DSBs. HR alone rescued cells from the lethal effects of thymidine. Furthermore, thymidine
increased the frequency of DNA exchange mediated solely by HR in the absence of detectable DSBs. Our data suggest that both
NHEJ and HR are involved in repair of arrested replication forks that include a DSB, while HR alone is required for the repair
of slowed replication forks in the absence of detectable DSBs.
We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities.
BCR/ABL regulates cell proliferation, apoptosis, differentiation and adhesion. In addition, BCR/ABL can induce resistance to cytostatic drugs and irradiation by modulation of DNA repair mechanisms, cell cycle checkpoints and Bcl-2 protein family members. Upon DNA damage BCR/ABL not only enhances reparation of DNA lesions (e.g. homologous recombination repair), but also prolongs activation of cell cycle checkpoints (e.g. G2/M) providing more time for repair of otherwise lethal lesions. Moreover, by modification of anti-apoptotic members of the Bcl-2 family (e.g. upregulation of Bcl-x(L)) BCR/ABL provides a cytoplasmic 'umbrella' protecting mitochondria from the 'rain' of apoptotic signals coming from the damaged DNA in the nucleus, thus preventing release of cytochrome c and activation of caspases. The unrepaired and/or aberrantly repaired (but not lethal) DNA lesions resulting from spontaneous and/or drug-induced damage can accumulate in BCR/ABL-transformed cells leading to genomic instability and malignant progression of the disease. Inhibition of BCR/ABL kinase activity by STI571 (Gleevec, imatinib mesylate) reverses drug resistance and, in combination with standard chemotherapeutics can exert strong anti-leukemia effect.
Camptothecin (CPT) is an anticancer drug that promotes DNA breakage at replication forks and the formation of lesions that
activate the processes of homologous recombination (HR) and nonhomologous end joining. We have taken advantage of the CPT-induced
damage response by coupling it to gene repair directed by synthetic oligonucleotides, a process in which a mutant base pair
is converted into a wild-type one. Here, we show that pretreating DLD-1 cells with CPT leads to a significant stimulation
in the frequency of correction of an integrated mutant enhanced green fluorescent protein gene. The stimulation is dose-dependent
and coincident with the formation of double-strand DNA breaks. Caffeine, but not vanillin, blocks the enhancement of gene
repair suggesting that, in this system, HR is the pathway most responsible for elevating the frequency of correction. The
involvement of HR is further proven by studies in which wortmannin was seen to inhibit gene repair at high concentrations
but not at lower levels that are known to inhibit DNA-PK activity. Taken together, our results suggest that DNA damage induced
by CPT activates a cellular response that stimulates gene repair in mammalian cells.
The Rad51 recombinase is an essential factor for homologous recombination and the repair of DNA double strand breaks, binding transiently to both single stranded and double stranded DNA during the recombination reaction. The use of a homologous recombination mechanism to repair DNA damage is controlled at several levels, including the binding of Rad51 to single stranded DNA to form the Rad51 nucleofilament, which is controlled through the action of DNA helicases that can counteract nucleofilament formation. Overexpression of Rad51 in different organisms and cell types has a wide assortment of consequences, ranging from increased homologous recombination and increased resistance to DNA damaging agents to disruption of the cell cycle and apoptotic cell death. Rad51 expression is increased in p53-negative cells, and since p53 is often mutated in tumor cells, there is a tendency for Rad51 to be overexpressed in tumor cells, leading to increased resistance to DNA damage and drugs used in chemotherapies. As cells with increased Rad51 levels are more resistant to DNA damage, there is a selection for tumor cells to have higher Rad51 levels. While increased Rad51 can provide drug resistance, it also leads to increased genomic instability and may contribute to carcinogenesis.
The mammalian Rad51 protein is involved in homologous recombination and in DNA damage repair. Its nuclear distribution after DNA damage is highly dynamic, and distinct foci of Rad51 protein, distributed throughout the nuclear volume, are induced within a few hours after γ irradiation; these foci then coalesce into larger clusters. Rad51-positive cells do not undergo DNA replication. Rad51 foci colocalize with both replication protein A and sites of unscheduled DNA repair synthesis and may represent a nuclear domain for recombinational DNA repair. By 24 h postirradiation, most foci are sequestered into micronuclei or assembled into Rad51-coated DNA fibers. These micronuclei and DNA fibers display genome fragmentation typical of apoptotic cell death. Other repair proteins, such as Rad52 and Gadd45, are not eliminated from the nucleus. DNA double strand breaks in repair-deficient cells or induced by the clastogen etoposide are also accompanied by the sequestering of Rad51 protein before cell death. The spindle poison colcemid causes cell cycle arrest and Rad51-foci formation without directly damaging DNA. Collectively, these observations suggest that mammalian Rad51 protein associates with damaged DNA and/or with DNA that is temporarily or irreversibly unable to replicate and these foci may subsequently be eliminated from the nucleus.
Rad51, of Saccharomyces cerevisiae, is a homologue of recA of Escherichia coli and plays crucial roles in both mitotic and meiotic recombination and in repair of double−strand breaks of DNA. We have cloned genes from human, mouse and fission yeast that are homologous to rad51. The 339 amino acid proteins predicted for the two mammalian genes are almost identical and are highly homologous (83%) with the yeast proteins. The mouse gene is transcribed at a high level in thymus, spleen, testis andpvary and at a lower level in brain and other tissues. The rad51 homologues fail to complement the DNA repair defect of rad51 mutants of S. cerevisiae. The mouse gene is located in the F1 region of chromosome 2 and the human gene maps to chromosome 15.
Isotype switching is the DNA recombination mechanism by which antibody genes diversify immunoglobulin effector functions. In contrast to V(D)J recombination, which is mediated by RAG1, RAG2 and DNA double-stranded break (DSB) repair proteins, little is known about the mechanism of switching. We have investigated the role of DNA DSB repair in switch recombination in mice that are unable to repair DSBs due to a deficiency in Ku80 (Ku80(-/-)). B-cell development is arrested at the pro-B cell stage in Ku80(-/-) mice because of abnormalities in V(D)J recombination, and there are no mature B cells. To reconstitute the B-cell compartment in Ku80(-/-) mice, pre-rearranged VB1-8 DJH2 (mu i) and V3-83JK2 (kappa i) genes were introduced into the Ku80(-/-) background (Ku80(-/-)mu i/+kappa i/+). Ku80(-/-)mu i/+ kappai/+ mice develop mature mIgM+ B cells that respond normally to lipopolysaccharide (LPS) or LPS plus interleukin-4 (IL-4) by producing specific germline Ig constant region transcripts and by forming switch region-specific DSBs. However, Ku80(-/-)mu i/+kappa i/+ B cells are unable to produce immunoglobulins of secondary isotypes, and fail to complete switch recombination. Thus, Ku80 is essential for switch recombination in vivo, suggesting a significant overlap between the molecular machinery that mediates DNA DSB repair, V(D)J recombination and isotype switching.
A sensitive and rapid in situ method was developed to visualize sites of single-stranded (ss) DNA in cultured cells and in experimental test animals. Anti-bromodeoxyuridine antibody recognizes the halogenated base analog incorporated into chromosomal DNA only when substituted DNA is in the single strand form. After treatment of cells with DNA-damaging agents or γ irradiation, ssDNA molecules form nuclear foci in a dose-dependent manner within 60 min. The mammalian recombination protein Rad51 and the replication protein A then accumulate at sites of ssDNA and form foci, suggesting that these are sites of recombinational DNA repair.
Rad51 protein of Saccharomyces cerevisiae is a structural homolog of the Escherichia coli recombination enzyme RecA. In yeast, the Rad51 protein is required for mitotic and meiotic recombination and for repair of double-strand breaks in DNA. We have used antibodies raised against the homologous human protein, HsRad51, expressed in E. coli, to visualize the spatial distribution of the protein in mammalian somatic and meiotic cells. In cultured human cells, the HsRad51 protein is concentrated in multiple discrete foci in the nucleoplasm; it is largely absent from cytoplasm and nucleoli. After treatment of cells with methyl methanesulfonate, ultraviolet irradiation, or 137Cs irradiation, the percentage of cells with HsRad51 protein immunofluorescence increases; the same cells show unscheduled DNA synthesis. Induction of Rad51 foci is blocked by inhibitors of transcription. In mouse pachytene spermatocytes, the mouse homolog of Rad51 protein is highly enriched in synaptonemal complexes that are formed between the paired homologous chromosomes during meiotic prophase. We conclude that the mammalian proteins homologous to yeast Rad51 are involved in repair of DNA damage and recombinational repair during meiosis.
The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability.
Rad51 is a highly conserved eukaryotic homolog of the prokaryotic recombination protein RecA, which has been shown to function in both recombinational repair of DNA damage and meiotic recombination in yeast. In primary murine B cells cultured with lipopolysaccharide (LPS) to stimulate heavy chain class switch recombination, Rad51 protein levels are dramatically induced. Immunofluorescent microscopy shows that anti-Rad51 antibodies stain foci that are localized within the nuclei of switching B cells. Immunohistochemical analysis of splenic sections shows that clusters of cells that stain brightly with anti-Rad51 antibodies are evident within several days after primary immunization and that Rad51 staining in vivo is confined to B cells that are switching from expression of IgM to IgG antibodies. Following switch recombination, B cells populate splenic germinal centers, where somatic hypermutation and clonal proliferation occur. Germinal center B cells are not stained by anti-Rad51 antibodies. Rad51 expression is therefore not coincident with somatic hypermutation, nor does Rad51 expression correlate simply with cell proliferation. These data suggest that Rad51, or a highly related member of the conserved RecA family, may function in class switch recombination.
RecA in Escherichia coli and its homolog, ScRad51 in Saccharomyces cerevisiae, are known to be essential for recombinational
repair. The homolog of RecA and ScRad51 in mice, MmRad51, was mutated to determine its function. Mutant embryos arrested early
during development. A decrease in cell proliferation, followed by programmed cell death and chromosome loss, was observed.
Radiation sensitivity was demonstrated in trophectoderm-derived cells. Interestingly, embryonic development progressed further
in a p53 null background; however, fibroblasts derived from double-mutant embryos failed to proliferate in tissue culture.
Chinese hamster lung fibroblast V79 cells have been widely used in studies of DNA damage and DNA repair. Since the p53 gene
is involved in normal responses to DNA damage, we have analyzed the molecular genetics and functional status of p53 in V79
cells and primary Chinese hamster embryonic fibroblast (CHEF) cells. The coding product of the p53 gene in CHEF cells was
76 and 75% homologous to human and mouse p53 respectively, and was 95% homologous to the Syrian hamster cells. The V79 p53
sequence contained two point mutations located within a presumed DNA binding domain, as compared with the CHEF cells. Additional
immunocytochemical and molecular studies confirmed that the p53 protein in V79 cells was mutated and nonfunctional. Our results
indicate that caution should be used in interpreting studies of DNA damage, DNA repair and apoptosis in V79 cells.
Normal diploid cells have a limited replicative potential in culture, with progressively increasing interdivision time. Rarely,
cell lines arise which can divide indefinitely; like tumor cells, such "immortal" lines display frequent chromosomal aberrations
which may reflect high rates of recombination. Recombination frequencies within a plasmid substrate were 3.5-fold higher in
nine immortal human cell lines than in six untransformed cell strains. Expression of HsRAD51, a human homolog of the yeast
RAD51 and Escherichia coli recA recombinase genes, was 4.5-fold higher in immortal cell lines than in mortal cells. Stable
transformation of human fibroblasts with simian virus 40 large T antigen prior to cell immortalization increased both chromosomal
recombination and the level of HsRAD51 transcripts by two- to fivefold. T-antigen induction of recombination was efficiently
blocked by introduction of HsRAD51 antisense (but not control) oligonucleotides spanning the initiation codon, implying that
HsRAD51 expression mediates augmented recombination. Since p53 binds and inactivates HsRAD51, T-antigen-p53 association may
block such inactivation and liberate HsRAD51. Upregulation of HsRAD51 transcripts in T-antigen-transformed and other immortal
cells suggests that recombinase activation can also occur at the RNA level and may facilitate cell transformation to immortality.
Yeast rad51 mutants are viable, but extremely sensitive to gamma-rays due to defective repair of double-strand breaks. In contrast, disruption of the murine RAD51 homologue is lethal, indicating an essential role of Rad51 in vertebrate cells. We generated clones of the chicken B lymphocyte line DT40 carrying a human RAD51 transgene under the control of a repressible promoter and subsequently disrupted the endogenous RAD51 loci. Upon inhibition of the RAD51 transgene, Rad51- cells accumulated in the G2/M phase of the cell cycle before dying. Chromosome analysis revealed that most metaphase-arrested Rad51- cells carried isochromatid-type breaks. In conclusion, Rad51 fulfils an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes.
Immunoglobulin (Ig) heavy chain (HC) class switch recombination (CSR) is a late B cell process that involves intrachromosomal DNA rearrangement. Ku70 and Ku80 form a DNA end-binding complex required for DNA double strand break repair and V(D)J recombination. Ku70(-/-) (K70T) mice, like recombination activating gene (RAG)-1- or RAG-2-deficient (R1T or R2T) mice, have impaired B and T cell development at an early progenitor stage, which is thought to result at least in part from defective V(D)J recombination (Gu, Y., K.J. Seidl, G.A. Rathbun, C. Zhu, J.P. Manis, N. van der Stoep, L. Davidson, H.L. Cheng, J.M. Sekiguchi, K. Frank, et al. 1997. Immunity. 7:653-665; Ouyang, H., A. Nussenzweig, A. Kurimasa, V.C. Soares, X. Li, C. Cordon-Cardo, W. Li, N. Cheong, M. Nussenzweig, G. Iliakis, et al. 1997. J. Exp. Med. 186:921-929). Therefore, to examine the potential role of Ku70 in CSR, we generated K70T mice that carry a germline Ig HC locus in which the JH region was replaced with a functionally rearranged VH(D)JH and Ig lambda light chain transgene (referred to as K70T/HL mice). Previously, we have shown that B cells from R1T or R2T mice carrying these rearranged Ig genes (R1T/HL or R2T/HL mice) can undergo CSR to IgG isotypes (Lansford, R., J. Manis, E. Sonoda, K. Rajewsky, and F. Alt. 1998. Int. Immunol. 10:325-332). K70T/HL mice had significant numbers of peripheral surface IgM+ B cells, which generated serum IgM levels similar to those of R2T/HL mice. However, in contrast to R2T/HL mice, K70T/HL mice had no detectable serum IgG isotypes. In vitro culture of K70T/HL B cells with agents that induce CSR in normal or R2T/HL B cells did lead to the induction of germline CH transcripts, indicating that initial signaling pathways for CSR were intact in K70T/HL cells. However, treatment with such agents did not lead to detectable CSR by K70T/HL B cells, and instead, led to cell death within 72 h. We conclude that Ku70 is required for the generation of B cells that have undergone Ig HC class switching. Potential roles for Ku70 in the CSR process are discussed.
A growing body of carcinogens are known to affect genetic recombination in mammalian cells and to thereby interfere with the process of carcinogenesis. In order to screen for recombinogenic effects of chemical and physical agents a variety of in vitro assay systems utilizing mammalian cells have been developed. However, the effects of potential carcinogens differ in these different systems. In order to investigate this phenomenon further, we have employed two different assay procedures, involving spontaneous duplication mutants in mammalian cells, which respond to homologous or non-homologous recombination. Four carcinogens were investigated, i.e. Aroclor 1221, benzene, methylmethanesulphonate (MMS) and thiourea, as were gamma- and UV-irradiation. With the exception of thiourea all of these factors resulted in elevated frequencies of homologous recombination. On the other hand, only UV-irradiation affected the rate of non-homologous recombination. These results indicate that substrate length and/or the recombination mechanism may influence the recombinogenic response of mammalian fibroblasts to carcinogenic factors. Thus, procedures for recombinogenic effects of carcinogens should consider the different pathways of recombination occurring in mammalian cells.
Etoposides block cell division by interfering with the action of topoisomerase II, leaving enzyme-DNA double-strand breaks. We found that certain components of the trimeric DNA-dependent protein kinase influence cell survival following etoposide damage. Interestingly, either Ku70- or Ku80-deficient cell lines, but not mutant cell lines of the DNA-PK catalytic sub-unit (DNA-PKcs), were found to be hypersensitive to the effects of etoposide VP16. Ku70- and Ku80-deficient cells can be complemented to an etoposide resistant phenotype by introducing wildtype Ku70 or Ku80 cDNAs. Mutational analysis of introduced Ku70 cDNAs into murine embryonic stem cells deleted for Ku70 (-/-) showed that mutants where heterodimerization and DNA binding functions of Ku were disrupted, also blocked the restoration of etoposide resistance. In contrast with the differential etoposide sensitivity of DNA-PK mutants, both Ku- and DNA-PKcs-deficient cell lines showed G2 ionizing radiation-induced delays, a cell cycle phase where topoisomerase II function is critical. Thus, the topoisomerase II cleaved complexes may be an example of DNA lesions requiring the Ku heterodimer, but not DNA-PK for DNA repair.
Ig heavy chain isotype switching in B lymphocytes is known to be preceded by transcription of a portion of the particular heavy chain gene segment that is targeted for recombination. Here, we describe an active role for these transcripts in the switch recombination process. Using an in vitro assay that exposes an artificial switch-mu (Smu) minisubstrate to switch region transcripts in the presence of nuclear extracts from switching cells, we demonstrate that free 3' ends of the Smu sequence are extended onto switch region transcripts by reverse transcription. The activity was induced in splenic B lymphocytes upon activation with LPS or CD40 ligand. This in vitro process is thought to be relevant to in vivo class switching for two reasons: 1) although only one-third of the Smu minisubstrate actually contains Smu sequence, all crossovers between switch regions occurred in the Smu portion; and 2) treatment of B lymphocytes with IL-4, which enriches for switching to S gamma 1, increases the ratio of Smu-S gamma 1 to Smu-S gamma 3 hybrids by 16% after LPS treatment and by 37% after CD40 ligand activation, implicating this S mu-primed reverse transcription of switch region transcripts as a novel mechanism of regulating the specificity of isotype switching. Further evidence for an active role of switch region transcripts was obtained by expressing S alpha RNA in trans in the Bcl1B1 B lymphoma line. Endogenous S mu-S alpha switch circles were detected in Bcl1B1 cells expressing exogenous S alpha RNA but not in mock-transfected cells.
The ideal therapy for single gene disorders would be repair of the mutated disease genes. Homologous recombination is one of several cellular mechanisms for the repair of DNA damage. Recombination between exogenous DNA and homologous chromosomal loci (gene targeting) can be used to repair an endogenous gene, but the low efficiency of this process is a serious barrier to its therapeutic potential. Recent progress in the isolation and characterisation of mammalian genes and proteins involved in DNA recombination has raised the possibility that the cellular biochemistry of recombination can be manipulated to improve the efficiency of gene targeting. As an initial test of this approach, we have overexpressed the gene encoding hRAD51, a protein with homologous DNA pairing and strand exchange activities, in human cells and measured its effect on gene targeting. We report a two- to three-fold increase in gene targeting, and enhanced resistance to ionising radiation in hRAD51-overexpressing cells with no obvious detrimental effects. These observations provide valuable genetic evidence for the involvement of hRAD51 in both gene targeting and DNA repair in human cells. Our data also establish overexpression of recombination genes as a viable approach to improving gene targeting efficiencies.
Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.
Information concerning the function of recombination proteins in mammalian cells has been obtained from biochemical studies, but little is known about their mechanisms of action in growing cells. The eukaryotic recombination protein RAD51, a homologue of the Escherichia coli RecA protein, has been shown to interact with various proteins, including the p53 protein, the guardian of genomic stability maintenance. Here, the hamster RAD51 protein, CgRAD51, has been overexpressed in the SPD8 cell line, derived from Chinese hamster V79 cells. This cell line offers unique possibilities for studying different mechanisms for homologous recombination on endogenous substrates. We report that the SPD8 cell line contains a mutated p53 gene, which provides new insights into the recombination process in these cells. The present study demonstrates that overexpression of CgRAD51 in these cells results in a two- to threefold increase in endogenous recombination. In addition, sequence analysis indicated that RAD51 promotes homologous recombination by a chromatid exchange mechanism.
RecA protein of E.coli plays a central regulatory role that is induced by damage to DNA and results in the inactivation of
LexA repressor. In vitro, RecA protein binds preferentially to single-stranded DNA to form a nucleoprotein filament that can
recognize homology in naked duplex DNA and promote extensive strand exchange. Although RecA protein shows little tendency
at neutral pH to bind to RNA, we found that It nonetheless catalyzed at 37°C the hybridization of complementary RNA and single-stranded DNA sequences. Hybrids made by RecA protein at 37°C appeared indistinguishable from ones prepared by thermal annealing. RNA-DNA hybridization by RecA protein at neutral pH
required, as does RecA-promoted homologous pairing, optimal conditions for the formation of RecA nucleoprotein filaments.
The cosedimentation of RNA with those filaments further paralleled observations made on the formation of networks of nucleoprotein
filaments with doublestranded
DNA, an Instrumental intermediate in homologous pairing In vitro. These similarities with the pairing reaction support the
view that RecA protein acts specifically in the hybridization reaction.
Recombination is a process thought to be underlying genomic instability involved in carcinogenesis. This report examines the potential of cytostatic drugs to induce intrachromosomal homologous recombination. In order to address this question, the hprt gene of a well-characterized mammalian cell line was employed as a unique endogenous marker for homologous recombination. Commonly used cytostatic drugs with different mode of action were investigated in this context, i.e. bifunctional alkylating agents, inhibitors of DNA synthesis, inhibitors of topoisomerases and a spindle poison. With the exception of the spindle poison, all these drugs were found to induce homologous recombination, with clear differences in their recombination potency, which could be related to their mechanism of action. Bifunctional alkylating agents were the least efficient, whereas inhibitors of DNA synthesis were found to be the most potent inducers of homologous recombination. This raises the question whether these later drugs should be considered for adverse effects in cancer chemotheraphy.
A total of 76 independent spontaneous mutants in the hprt gene of V79 Chinese hamster cells have been analyzed. These mutants were obtained in two different laboratories, 17 and 59 mutants in sets 1 and 2, respectively, under different cell culture conditions. Mutation analysis was performed by amplification of hprt cDNA with the polymerase chain reaction and direct sequencing of the products. The data obtained showed similar spectra of spontaneous mutations in both sets of mutants, suggesting that culture does not play a major role in spontaneous mutagenesis. The majority of the mutations were base substitutions (>60%), with twice as many transversions as transitions. Base changes were evenly distributed throughout the structural gene, including the splice junctions. All types of base substitutions appeared in comparable frequencies, except for A · T to T · A transversions, which were almost absent. The fraction of deletion mutations was low (13%). A striking feature of the observed mutation spectra is that one third of the spontaneous mutations analyzed involved aberrant splicing of the hprt primary transcript, with exon 4 being affected most frequently, indicating that splice mutations are a common mechanism of mutation in the hprt gene.
Here, the sequence in the hprt gene of the duplication mutant SPD8 originating from V79 Chinese hamster cells was determined. The duplication arose after non-homologous recombination between exon 6 and intron 7, resulting in an extra copy of the 3′ portion of exon 6, of exon 7 and of flanking intron regions. Only a duplication of exon 7 is present in the mRNA, since the duplicated exon 6 lacks its 5′ splice site and is removed during RNA processing. The findings in this study suggest that the non-homologous recombination mechanism which occurred here may have been initiated by endonucleases, rather than by a spontaneous double strand break. Subsequently, 14 spontaneous SPD8 revertants with a functional hprt gene were isolated and characterized using PCR and sequencing. The data revealed that although the SPD8 cell line arose by non-homologous recombination, it reverts spontaneously by homologous recombination. Interestingly, the downstream copy of exon 7 was restored by this process. This was indicated by the presence of a specific mutation, a T-to-G transversion, close to the breakpoint, a characteristic unique to the SPD8 clone. Our results suggest that the spontaneous reversion of this cell line by homologous recombination may involve an exchange, rather than a conversion mechanism.
RecA protein of E. coli plays a central regulatory role that is induced by damage to DNA and results in the inactivation of LexA repressor. In vitro, RecA protein binds preferentially to single-stranded DNA to form a nucleoprotein filament that can recognize homology in naked duplex DNA and promote extensive strand exchange. Although RecA protein shows little tendency at neutral pH to bind to RNA, we found that it nonetheless catalyzed at 37 degrees C the hybridization of complementary RNA and single-stranded DNA sequences. Hybrids made by RecA protein at 37 degrees C appeared indistinguishable from ones prepared by thermal annealing. RNA-DNA hybridization by RecA protein at neutral pH required, as does RecA-promoted homologous pairing, optimal conditions for the formation of RecA nucleoprotein filaments. The cosedimentation of RNA with those filaments further paralleled observations made on the formation of networks of nucleoprotein filaments with double-stranded DNA, an instrumental intermediate in homologous pairing in vitro. These similarities with the pairing reaction support the view that RecA protein acts specifically in the hybridization reaction.
The nucleoprotein filament formed by the RecA protein of Escherichla coll on single-stranded DNA catalyzes the hybridization
of RNA transcripts with singlestranded DNA sequences at 37°C, In vitro. RecA protein rapidly promotes hybridization, even when noncomplementary RNA is In a milllonfold nucleotide excess
over hybridizing RNA, and in a thousandfold nucleotide excess over hybridizing single-stranded DNA. Heterologous double-stranded
DNA and RecAcoated noncomplementary single-stranded DNA are also poor competitors of RNA transcripts produced in vitro . Since large excesses of noncomplementary RNA fail to inhibit sharply the hybridization reaction by RecA protein under mild,
non-degradative conditions, the reaction may be useful in the identification and isolation of transcripts produced In vivo.
The spontaneous hprt mutant clone SP5, derived from V79 Chinese hamster cells, was shown to exhibit a duplication of approximately 2 kb, including exon 2 and its flanking intron sequences, inserted into the intron 1 sequence of the hprt gene. The most striking feature of SP5 is that this clone is quite unstable, demonstrating an extremely high spontaneous reversion frequency. Molecular analysis of 25 independent revertant clones of SP5 indicated that they arose after precise deletion of the duplicated fragment in the hprt gene. Reversion of SP5 could be induced by agents which damage DNA by different mechanisms, but there was no correlation with induction of the forward mutations. Based on these results, we suggest that intrachromosomal recombination must be responsible for the spontaneous reversion of SP5. Genetic recombination in somatic cells has been suggested to be involved in the multistep process of carcinogenesis. Since the ability to induce intrachromosomal recombination in yeast has been shown to be highly correlated with non-mutagenic as well as mutagenic carcinogens, it is of great interest to investigate similar systems in mammalian cells. The SP5 cell line may be unique for such a purpose, since this mutant clone contains an endogenic marker for studying the process of intrachromosomal recombination.
A deletion DNA rearrangement is associated with immunoglobulin class switching from IgM to IgG, IgA or IgE This recombination occurs in immunoglobulin switch regions, which are complex, highly repetitive regions of DNA. As switch regions become transcriptionally active just before switch recombination, analysis of the behaviour of these sequences during transcription could elucidate the mechanism of switch recombination. Here, we report that transcription of a supercoiled plasmid containing the murine IgA switch region (S alpha) leads to a loss of superhelical turns. The resulting series of less supercoiled plasmids is stabilized by RNA-DNA hybrids formed by the nascent RNA transcripts, which remain base-paired with their DNA templates.
Murine cells homozygous for the severe combined immune deficiency mutation (scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PKcs, the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PKcs gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PKcs is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.
In the present study 34 agents, related to carcinogenesis in different ways, were investigated with respect to their recombinogenic activity in mammalian cells. The induction of intrachromosomal recombination was studied using the spontaneous mutant clone SP5 derived from V79 Chinese hamster cells, which exhibits a duplication of exon 2 and its flanking regions in the hprt gene, which was found to be inserted between the two EcoR1 sites of intron 1. Earlier studies on the removal of this insertion fragment in the SP5 clone indicated that such loss involved intrachromosomal recombination and was detectable by using a reversion mutation assay. The categories of agents investigated here included monofunctional alkylating agents, polyaromatic hydrocarbons giving rise to bulky adducts, chlorinated compounds giving small cyclic adducts, intercalating agents, DNA cross-linkers, UV and ionizing radiation, inhibitors of DNA synthesis and topoisomerases, DNA bases and base analogues, radical formers and tumour promotors. Statistically significant enhancements in the frequency of reversion in SP5 cells were observed after treatment with aflatoxin B1, 9-aminoacridine, benzo[a]-pyrene-7,8-dihydrodiol, benzo[a]pyrene-7,8-diol-9,10-epoxide, camptothecin, dimethylbenzanthracene, dimethyl-nitrosamine, ethidium bromide, ethylmethanesulfonate, N-ethyl-N'-nitrosourea, fluorodeoxyuridine, ICR 191, N-methyl-N'-nitrosoguanidine, mitomycin C and UV irradiation. Only slight inducing effects were indicated in the case of methylmethanesulfonate, N-methyl-N'-nitrosourea and gamma irradiation, although not statistically significant. Negative results were found after treatment with 3-amino-benzamide, 5-azacytidine, bleomycin, 5-bromodeoxyuridine, 1,2-dichloroethane, ethylene oxide, etoposide, formaldehyde, hydrogen peroxide, methotrexate, propylene oxide, quercitin, sodium azide, 12-O-tetradecanoylphorbol-13-acetate, thymidine and a complex mixture consisting of a cigarette smoke condensate. Our results on chemically or physically induced recombinogenic effects in the endogenous SP5 hprt gene are in agreement with data obtained in non-endogenous gene systems based on transgenic cell lines containing integrates of tandem mutated tk or hygromycin resistance genes, but not completely consistent with findings on integrates based on the neo genes. This suggests that many factors influence the recombination process, including a difference in the mechanisms underlying inter- and intrachromosomal recombination. Consequently, the endogenous SP5/V79 system is suggested to be more representative than integrated systems for investigating induction of recombination, as well as for mechanistic studies of recombination at the molecular level, e.g. intrachromosomal recombination.
The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cells, is defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. The human XRCC5 DNA repair gene, which complements this mutant, is shown here through genetic and biochemical evidence to be the 80-kilodalton subunit of the Ku protein. Ku binds to free double-stranded DNA ends and is the DNA-binding component of the DNA-dependent protein kinase. Thus, the Ku protein is involved in DNA repair and in V(D)J recombination, and these results may also indicate a role for the Ku-DNA-dependent protein kinase complex in those same processes.
Analysis of mitotic and meiotic recombination in mammalian cells has been hampered by the complexity of the reactions involved as well as lack of mutants. Furthermore, none of the genes involved in the process has yet been identified. In budding yeast, Saccharomyces cerevisiae, the RAD51 gene is essential along with other genes of the RAD52 epistasis group for mitotic and meiotic recombination and DNA repair. The Rad51 protein is structurally similar to Escherichia coli RecA protein, which is required in homologous recombination and SOS responses in bacteria. Here we report the isolation of a mouse homolog of the yeast RAD51 gene. The amino acid sequence predicted from the gene shows 83% and 55% homology with those of the yeast RAD51 and the E. coli recA product, respectively. The mouse gene complemented a rad51 mutation of S. cerevisiae with sensitivity to methyl-methanesulfonate, which produces double-strand breaks of DNA. This gene is expressed in the thymus, testis, ovary, spleen, and intestine, suggesting that its product is involved in mitotic and meiotic recombination in addition to DNA repair.
Sequencing of hprt cDNA revealed that three spontaneous mutants in V79 Chinese hamster cells exhibit tandem duplications of exon(s), i.e., either exons 2 and 3 or exon 7. Sequences of different sizes (4.5-8 Kb) were found to be duplicated and inserted in tandem into the hprt gene. These mutants demonstrated spontaneous reversion frequencies which were about 40-fold higher than those observed with other types of spontaneous mutants, but on the same order of magnitude as spontaneous reversions in Sp5, a mutant with a duplication insertion involving exon 2 in this gene. These data suggest that all of the duplications found have the same genetic instability, regardless of the type, size or position of the duplicated fragment. The coding sequence of the hprt cDNA and the restriction pattern of the revertants were virtually identical to the wild-type, indicating restoration of a functional hprt gene by precise deletion of the duplicated fragment.
The human testis Rad51 protein, a structural homolog of E. coli RecA, binds single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using circular ssDNA and linear dsDNA (3.0 kb in length), we demonstrate that hRad51 promotes homologous pairing and strand exchange reactions in vitro. Joint molecule formation was dependent upon ATP hydrolysis and DNA homology and was stimulated by the single-strand DNA-binding protein RP-A. In these reactions, the 5' terminus of the complementary strand of the linear duplex was efficiently transferred to the ssDNA. However, under standard conditions, extensive strand exchange was not observed. These results establish hRad51 as a functional homolog of RecA, but indicate that the human protein and its bacterial counterpart differ in their ability to promote extensive strand transfer. It is proposed that hRad51 mediates homology recognition and initiates strand exchange, but that extensive heteroduplex formation in higher organisms requires the actions of additional proteins.
Lipopolysaccharide (LPS) is a B cell mitogen which can stimulate murine primary B cells to proliferate and carry out immunoglobulin heavy chain class switch recombination. LPS can also function as an endotoxin, which may cause DNA damage and apoptosis in certain types of cells. We have previously reported that LPS-activated primary murine B cells contain nuclear foci that stain brightly with anti-Rad51 antibodies (Li et al. (1996) Proc. Natl. Acad. Sci. USA 93, 10222-10227). We have now analyzed Rad51 nuclear foci induced in both primary and immortalized B cells by treatment with the DNA damaging agent, methyl methanesulfonate (MMS). We have found that, in LPS-cultured primary B cells, MMS treatment increases the fraction of cells containing Rad51 foci and induces formation of a very high number of foci per cell. The foci induced by MMS treatment are small, punctate, and numerous; in contrast, the foci induced by LPS activation are large, brightly staining, and relatively few in number. In LPS-cultured primary B cells, Rad51 relocalizes during the cell cycle, and large, brightly staining nuclear foci are present in only restricted stages of the cell cycle. Rad51 foci similar to those present in LPS-activated primary B cells are also observed in immortalized B cells lines cultured in the absence of LPS. These foci are unaltered in number or appearance by culture with LPS, but treatment of immortalized B cell lines with MMS induces foci which are small and punctate in staining, like those induced by MMS in primary B cells. These data show that distinctive Rad51 foci are induced by DNA damaging agents and cell activation and that the response to DNA damage may involve pathways distinct from those associated with B cell activation and switch recombination.
Rad51 proteins share both structural and functional homologies with the bacterial recombinase RecA. The human Rad51 (HsRad51)
is able to catalyse strand exchange between homologous DNA molecules in vitro. However the biological functions of Rad51 in mammals are largely unknown. In order to address this question, we have cloned
hamster Rad51 cDNA and overexpressed the corresponding protein in CHO cells. We found that 2–3-fold overexpression of the
protein stimulated the homologous recombination between integrated genes by 20-fold indicating that Rad51 is a functional
and key enzyme of an intrachromosomal recombination pathway. Cells overexpressing Rad51 were resistant to ionizing radiation
when irradiated in late S/G2 phase of the cell cycle. This suggests that Rad51 participate in the repair of double-strand breaks most likely by homologous
recombination involving sister chromatids formed after the S phase.
Eukaryotic cells possess several mechanisms for repairing double-stranded breaks in DNA. One mechanism involves genetic recombination with an intact sister duplex. The recent identification of the RAD51 protein, a eukaryotic homologue of Escherichia coli RecA, represents a landmark discovery in our understanding of the key reactions in this repair pathway. RAD51 is similar to RecA, both biochemically and structurally: it promotes homologous pairing and strand exchange within a regular nucleoprotein filament. The isolation of yeast and human RecA homologues shows that homologous recombination and recombinational repair have been conserved throughout evolution. The goal is now to identify other factors involved in recombinational repair and to define their roles in this essential process.
Unlabelled:
Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation. DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity. The DNA-PK catalytic subunit (DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA-PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase-DNA cleavage complex could lead to a juxtaposition of replication fork-associated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S-phase checkpoint mechanism.
Keywords:
camptothecin/DNA damage/DNA-dependent protein kinase/RPA2 phosphorylation
Although it is clear that mammalian somatic cells possess the enzymatic machinery to perform homologous recombination of DNA molecules, the importance of this process in mitigating DNA damage has been uncertain. An initial genetic framework for studying homologous recombinational repair (HRR) has come from identifying relevant genes by homology or by their ability to correct mutants whose phenotypes are suggestive of recombinational defects. While yeast has been an invaluable guide, higher eukaryotes diverge in the details and complexity of HRR. For eliminating DSBs, HRR and end-joining pathways share the burden, with HRR contributing critically during S and G2 phases. It is likely that the removal of interstrand cross-links is absolutely dependent on efficient HRR, as suggested by the extraordinary sensitivity of the ercc1, xpf/ercc4, xrcc2, and xrcc3 mutants to cross-linking chemicals. Similarly, chromosome stability in untreated cells requires intact HRR, which may eliminate DSBs arising during DNA replication and thereby prevent chromosome aberrations. Complex regulation of HRR by cell cycle checkpoint and surveillance functions is suggested not only by direct interactions between human Rad51 and p53, c-Abl, and BRCA2, but also by very high recombination rates in p53-deficient cells.
Human cells can process DNA double-strand breaks (DSBs) by either homology directed or non-homologous repair pathways. Defects in components of DSB repair pathways are associated with a predisposition to cancer. The products of the BRCA1 and BRCA2 genes, which normally confer protection against breast cancer, are involved in homology-directed DSB repair. Defects in another homology-directed pathway, single-strand annealing, are associated with genome instability and cancer predisposition in the Nijmegen breakage syndrome and a radiation-sensitive ataxia-telangiectasia-like syndrome. Many DSB repair proteins also participate in the signaling pathways which underlie the cell's response to DSBs.
DNA double-strand breaks (DSB) are considered to be critical primary lesions in the formation of chromosomal aberrations. DSB may be induced by exogenous agents, such as ionizing radiation, but also occur spontaneously during cellular processes at quite significant frequencies. To repair this potentially lethal damage, eukaryotic cells have evolved a variety of repair pathways related to homologous and illegitimate recombination, also called non-homologous DNA end joining, which may induce small scale mutations and chromosomal aberrations. In this paper we review the major cellular sources of spontaneous DSB and the different homologous and illegitimate recombination repair pathways, with particular focus on their potential to induce chromosomal aberrations.
In mammalian cells, double-strand breaks in DNA can be repaired by nonhomologous end-joining (NHEJ), a process dependent upon Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV. Starting with HeLa cell-free extracts, which promote NHEJ in a reaction dependent upon all of these proteins, we have purified a novel factor that stimulates DNA end-joining in vitro. Using a combination of phosphorus NMR, mass spectroscopy, and strong anion exchange chromatography, we identify this factor as inositol hexakisphosphate (IP6). Purified IP6 is bound by DNA-PK and specifically stimulates DNA-PK-dependent end-joining in vitro. The involvement of inositol phosphate in DNA-PK-dependent NHEJ is of particular interest since the catalytic domain of DNA-PKcs is similar to that found in the phosphatidylinositol 3 (PI 3)-kinase family.