![HR-deficient irs1SF cell line is sensitive to the toxic effects of both hydroxyurea and thymidine. Colony outgrowth of the Chinese hamster cell lines AA8 (wild type), irs1SF (deficient in HR protein XRCC3 [35]), CXR3 (irs1SF complemented with human XRCC3 [48]), and V3-3 (deficient in the NHEJ protein DNA-PKcs [8]) upon exposure to hydroxyurea (A) and thymidine (B) was determined. The means (symbols) and standard deviation (bars) of at least three experiments are shown.](https://www.researchgate.net/profile/Klaus-Erixon/publication/11244123/figure/fig4/AS:668199163011072@1536322503201/HR-deficient-irs1SF-cell-line-is-sensitive-to-the-toxic-effects-of-both-hydroxyurea-and_Q320.jpg)
![Hydroxyurea-induced DSBs are formed in newly replicated DNA. The DNA of AA8 cells was labeled with [ 14 C]thymidine ( 14 C-TdR), either homogeneously for 24 h or speci fi cally at sites of replication for 30 min, prior to exposure to hydroxyurea or ␥ -rays. DNA was separated by pulsed- fi eld gel electrophoresis and visualized by ethidium bromide staining (A) and autoradiography (B). The size distributions of DNA fragments released from AA8 cells labeled for 24 h (C) or 30 min (D) with [ 14 C]thymidine and subsequently treated with hydroxyurea (1 mM; black line) or ␥ -rays (100 Gy; gray line) were also determined, and 42% Ϯ 2% and 51% Ϯ 5% of total [ 14 C]thymidine-labeled DNA from 24-h- and 30-min-labeled cells, respectively, were released from the inserts by hydroxyurea treatment. The corresponding values for ␥ -ray treatments were 73% Ϯ 4% (24 h) and 29% Ϯ 3% (30 min). (E) Size distribution of DNA fragments with 3 H activity released from inserts containing cells labeled with [ 3 H]thymidine for 30 min and subsequently treated with hydroxyurea (1 mM).](https://www.researchgate.net/profile/Klaus-Erixon/publication/11244123/figure/fig3/AS:282322856366083@1444322422238/Hydroxyurea-induced-DSBs-are-formed-in-newly-replicated-DNA-The-DNA-of-AA8-cells-was_Q320.jpg)
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Different Roles for Nonhomologous End Joining and Homologous Recombination following Replication Arrest in Mammalian Cells
Abstract and Figures
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.
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... In this pathway, HR exploits the duplicate genetic data located on the homologous sister chromatid. During the S-phase of DNA replication HR uses the sister chromatid as a reference to restore nucleotides of damaged DNA in the instance of replication fork stalling [44,45]. BRCA1 promotes end resection with BRCA2 loading RAD51, which in turn facilitates error free repair by catalyzing the homology search on the sister chromatid [46]. ...
... In this pathway, HR exploits the duplicate ge netic data located on the homologous sister chromatid. During the S-phase of DNA repli cation HR uses the sister chromatid as a reference to restore nucleotides of damaged DNA in the instance of replication fork stalling [44,45]. BRCA1 promotes end resection with BRCA2 loading RAD51, which in turn facilitates error free repair by catalyzing the homol ogy search on the sister chromatid [46]. ...
Since 2010, significant progress has been made in the treatment of metastatic castrate resistant prostate cancer (mCRPC). While these advancements have improved survival, mCRPC remains a lethal disease, with a precision medicine framework that is lagging behind compared to other cancers. Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) studies in prostate cancer (PCa) have focused primarily on the homologous recombination repair (HRR) genes, specifically BRCA1 and BRCA2. While homologous recombination deficiency (HRD) can be prompted by germline or somatic BRCA1/2 genetic mutations, it can also exist in tumors with intact BRCA1/BRCA2 genes. While the sensitivity of PARPi in tumors with non-BRCA DNA damage signatures is not as well established, it has been suggested that genomic alterations in DNA damage repair (DDR) genes other than BRCA may confer synthetic lethality with PARPI in mCRPC. The aim of this review is to summarize the literature on PARPi and their activity treating BRCA and non BRCA tumors with DNA damage signatures.
... Remarkably, the interaction between TIP60 and SET7 was significantly increased in response to DNA damage by HU (Supplementary Fig. 4C). HU, which is primarily dynamic in the S phase of the cell cycle, induces HR in mammalian cells by inhibiting replication (16,17). Additionally, HR and SET7 are associated with DDR by catalyzing the methylation of DDR protein ARTD1, and HR occurs during the S and G2 phases of the cell cycle (12,18). ...
... Substances such as HU, thymidine, and camptothecin induce replication fork collapse and strongly induce HR in mammalian cells. These substances require HR for cell survival (17). UHRF1 methylation by SET7 is necessary for cell survival in response to UV exposure (13). ...
The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is crucial for maintaining genomic integrity and is involved in numerous fundamental biological processes. Post-translational modifications by proteins play an important role in regulating DNA repair. Here, we report that the methyltransferase SET7 regulates HR-mediated DSB repair by methylating TIP60, a histone acetyltransferase and tumor suppressor involved in gene expression and protein stability. We show that SET7 targets TIP60 for methylation at K137, which facilitates DSB repair by promoting HR and determines cell viability against DNA damage. Interestingly, TIP60 demethylation is catalyzed by LSD1, which affects HR efficiency. Taken together, our findings reveal the importance of TIP60 methylation status by SET7 and LSD1 in the DSB repair pathway.
... The SSBs are restored by nucleotide excision repair, base excision repair, and base mismatch repair, whereas DSBs are repaired by HRR and nonhomologous end joining (4). During the S-phase of the cell cycle, the DNA replication phase, homologous recombination (HR) uses sister chromatids as a reference to restore the nucleotides damaged in the replication fork arrest (5). Therefore, targeting the DDR process may provide valuable therapeutic options for certain conditions promoting carcinogenesis through DDR gene mutation. ...
Prostate cancer (PC) is one of the major health issues of elderly men in the word. It is showed that there were approximately 1.414 million patients with PC in 2020 worldwide, with a high mortality rate in metastatic cases. In the present choices of treatment in PC, androgen deprivation therapy has long been as a backbone of them. But the clinical outcomes of patients with metastatic castration-resistant prostate cancer (mCRPC) were not ideal because of their poor prognosis, more effective therapeutic approaches are still necessary to further improve this problem. Poly (ADP-ribose) polymerase (PARP) inhibitors lead to the single-strand DNA breaks and/or double-strand DNA breaks, and result in synthetic lethality in cancer cells with impaired homologous recombination genes. It is estimated that approximately 20~25% of patients with mCRPC have a somatic or germinal DNA damage repair gene mutation. Furthermore, in “ BRCA ness” cases, which has been used to describe as tumors that have not arisen from a germline BRCA1 or BRCA2 mutation, there were also a number of studies sought to extend these promising results of PARP inhibitors. It is worth noting that an interaction between androgen receptor signaling and synthetic lethality with PARP inhibitors has been proposed. In this review, we discussed the mechanism of action and clinical research of PARP inhibitors, which may benefit population from “specific” to the “all-comer” in patients with PC when combined with novel hormonal therapies.
... Mounting evidence supports the notion that HR factors play key roles in the maintenance of genome stability in the response to DNA replication stress. Addition of DNA replication inhibitors results in an accumulation of nuclear foci of HR factors, enhancement of HR frequency in the genome, and an increase in growth defects in HRdeficient cells (Arnaudeau et al., 2000;Saintigny et al., 2001;Lundin et al., 2002). When DSBs are induced such that one of the two DSB ends has homology elsewhere in the genome, these DSBs are repaired by break-induced replication, which depends on HR factors (Anand et al., 2013). ...
Organisms have evolved elaborate mechanisms that maintain genome stability. Deficiencies in these mechanisms result in changes to the nucleotide sequence as well as copy number and structural variations in the genome. Genome instability has been implicated in numerous human diseases. However, genomic alterations can also be beneficial as they are an essential part of the evolutionary process. Organisms sometimes program genomic changes that drive genetic and phenotypic diversity. Therefore, genome alterations can have both positive and negative impacts on cellular growth and functions, which underscores the need to control the processes that restrict or induce such changes to the genome. The ribosomal RNA gene (rDNA) is highly abundant in eukaryotic genomes, forming a cluster where numerous rDNA copies are tandemly arrayed. Budding yeast can alter the stability of its rDNA cluster by changing the rDNA copy number within the cluster or by producing extrachromosomal rDNA circles. Here, we review the mechanisms that regulate the stability of the budding yeast rDNA cluster during repair of DNA double-strand breaks that are formed in response to programmed DNA replication fork arrest.
... HR utilizes the identical genetic information found on the homologous sister chromatid as a temporary template to replace nucleotides of damaged DNA in the event of replication fork stalling during S-phase DNA replication [58]. A series of recruited molecules orchestrate invasion of the sister chromatid by separating the two strands, polymerizing a copy of one template strand, excising the damaged region of DNA from the original chromatid, and inserting the copied genetic material (Figure 2) [59]. ...
BRCA1 and BRCA2 are tumor suppressor genes with pivotal roles in the development of breast and ovarian cancers. These genes are essential for DNA double-strand break repair via homologous recombination (HR), which is a virtually error-free DNA repair mechanism. Following BRCA1 or BRCA2 mutations, HR is compromised, forcing cells to adopt alternative error-prone repair pathways that often result in tumorigenesis. Synthetic lethality refers to cell death caused by simultaneous perturbations of two genes while change of any one of them alone is nonlethal. Therefore, synthetic lethality can be instrumental in identifying new therapeutic targets for BRCA1/2 mutations. PARP is an established synthetic lethal partner of the BRCA genes. Its role is imperative in the single-strand break DNA repair system. Recently, Olaparib (a PARP inhibitor) was approved for treatment of BRCA1/2 breast and ovarian cancer as the first successful synthetic lethality-based therapy, showing considerable success in the development of effective targeted cancer therapeutics. Nevertheless, the possibility of drug resistance to targeted cancer therapy based on synthetic lethality necessitates the development of additional therapeutic options. This literature review addresses cancer predisposition genes, including BRCA1, BRCA2, and PALB2, synthetic lethality in the context of DNA repair machinery, as well as available treatment options.
... NHEJ is a rapid repair process, and HR is known to be relatively slower (41,42). While MMEJ/TMEJ has previously been shown to be slower than NHEJ (8), its kinetics relative to HR remain unknown. ...
Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays ('PathSig-dPCR') for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.
... Given that hSSB1 S134 may therefore be phosphorylated in the response to these lesions, it is also interesting to consider the pathway through which this occurs. Indeed, whilst double-strand DNA breaks caused by fork collapse have traditionally been thought to be repaired solely by homologous recombination (HR) (Arnaudeau et al., 2001;Lundin et al., 2002), this has been challenged by the implication of numerous nonhomologous end-joining (NHEJ)-related proteins (e.g. DNA-PK, KU, Artmeis, DNA ligase IV) in the replication stress response (Allen et al., 2011). ...
... In the case of aphidicolin, it is a potent inhibitor of polymerases α, δ and ε, which leads to the blockage of the replication fork and provokes a similar effect to hydroxyurea (Wang, 1991). The effect on replication caused by these agents leads to double-strand DNA breaks (DSBs), which can stimulate both the HDR and the non-homologous end joining (NHEJ) pathways to repair the DNA damage (Lundin et al., 2002). Accordingly, the incubation of both DG44 and DG44-p11Mut cell lines with 5 µg/mL aphidicolin or 2 mM hydroxyurea for 3 h before incubation with the repair-PPRHs increased the repair frequency by 2-fold (Solé et al., 2014). ...
Monogenic disorders are often the result of single point mutations in specific genes, leading to the production of non-functional proteins. Different blood disorders such as ß-thalassemia, sickle cell disease, hereditary spherocytosis, Fanconi anemia, and Hemophilia A and B are usually caused by point mutations. Gene editing tools including TALENs, ZFNs, or CRISPR/Cas platforms have been developed to correct mutations responsible for different diseases. However, alternative molecular tools such as triplex-forming oligonucleotides and their derivatives (e.g., peptide nucleic acids), not relying on nuclease activity, have also demonstrated their ability to correct mutations in the DNA. Here, we review the Repair-PolyPurine Reverse Hoogsteen hairpins (PPRHs) technology, which can represent an alternative gene editing tool within this field. Repair-PPRHs are non-modified single-stranded DNA molecules formed by two polypurine mirror repeat sequences linked by a five-thymidine bridge, followed by an extended sequence at one end of the molecule which is homologous to the DNA sequence to be repaired but containing the corrected nucleotide. The two polypurine arms of the PPRH are bound by intramolecular reverse-Hoogsteen bonds between the purines, thus forming a hairpin structure. This hairpin core binds to polypyrimidine tracts located relatively near the target mutation in the dsDNA in a sequence-specific manner by Watson-Crick bonds, thus producing a triplex structure which stimulates recombination. This technology has been successfully employed to repair a collection of mutants of the dhfr and aprt genes within their endogenous loci in mammalian cells and could be suitable for the correction of mutations responsible for blood disorders.
KU70 (XRCC6 gene in humans) is one of the proteins in the KU70-KU80 heterodimer which is the first component recruited to broken DNA ends during DNA double-strand break repair through nonhomologous end joining (NHEJ). Previous studies have shown that Ku70 deficient mouse cells are defective in NHEJ and V(D)J recombination. In contrast, heterozygous KU70 mutant human cell lines did not show any significant change in cell viability and sensitivity towards ionizing radiation. In this study, we have used CRISPR-Cas9 technique to generate a KU70 mutant (heterozygous) human pre-B leukemic cell line (N6-KU70–2-DG). We observed that the N6-KU70–2-DG cells showed a prominent reduction in the expression of both KU70 mRNA and protein. The mutant cells showed reduced cell viability, increased sensitivity to DSB inducing agents such as ionizing radiation (IR) and etoposide, and increased number of unrepaired DSBs after exposure to IR. In addition, the mutant cells showed a reduction in the NHEJ activity and increased rate of microhomology mediated joining (MMEJ) activity. KU70 mutant cells also revealed enhanced level of senescence markers following irradiation. Thus, we report a novel KU70-mutant leukemic cell line (heterozygous) with reduced NHEJ, which is sensitive to DNA damaging agents, unlike the previously reported other KU heterozygous mutant cell lines.
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.
A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.
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.
Ionizing radiation and interstrand DNA crosslinking compounds provide important treatments against cancer due to their extreme genotoxicity for proliferating cells. Both the efficacies of such treatments and the mutagenic potential of these agents are modulated by the ability of cells to repair the inflicted DNA damage. Here we demonstrate that homologous recombination-deficient mRAD54-/- mice are hypersensitive to ionizing radiation at the embryonic but, unexpectedly, not at the adult stage. However, at the adult stage mRAD54 deficiency dramatically aggravates the ionizing radiation sensitivity of severe combined immune deficiency (scid) mice that are impaired in DNA double-strand break repair through DNA end-joining. In contrast, regardless of developmental stage, mRAD54-/- mice are hypersensitive to the interstrand DNA crosslinking compound mitomycin C. These results demonstrate that the two major DNA double-strand break repair pathways in mammals have overlapping as well as specialized roles, and that the relative contribution of these pathways towards repair of ionizing radiation-induced DNA damage changes during development of the animal.
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.
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.