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Pathways of DNA double-strand break repair by homologous recombination. Double-strand breaks (DSBs) can be repaired by distinctive homologous recombination (HR) pathways, such as synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR). (a) After DSB formation, the DNA ends are resected to yield 3′ single-strand DNA (ssDNA) overhangs, which become the substrate for the HR protein machinery to execute strand invasion of a partner chromosome. After a successful homology search, strand invasion occurs to form a nascent D-loop structure. DNA synthesis then ensues. (b) In the SDSA pathway, the D loop is unwound and the freed ssDNA strand anneals with the complementary ssDNA strand that is associated with the other DSB end. The reaction is completed by gap-filling DNA synthesis and ligation. Only noncrossover products are formed. (c) Alternatively, the second DSB end can be captured to form an intermediate that harbors two Holliday junctions (HJ)s, accompanied by gap-filling DNA synthesis and ligation. The resolution of HJs by a specialized endonuclease can result in either noncrossover (black triangles) or crossover products (gray triangles).
Source publication
Homologous recombination (HR) serves to eliminate deleterious lesions, such as double-stranded breaks and interstrand crosslinks, from chromosomes. HR is also critical for the preservation of replication forks, for telomere maintenance, and chromosome segregation in meiosis I. As such, HR is indispensable for the maintenance of genome integrity and...
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Homologous recombination is a major pathway for the repair of DNA double strand breaks, essential both to maintain genomic integrity and to generate genetic diversity. Mechanistically, homologous recombination involves the use of a homologous DNA molecule as a template to repair the break. In eukaryotes, the search for and invasion of the homologou...
Brh2, the ortholog of the BRCA2 tumor suppressor in Ustilago maydis, works hand in hand with Rad51 to promote repair of DNA by homologous recombination. Previous studies established that Brh2 can stimulate DNA strand exchange by enabling Rad51 nucleoprotein filament formation on replication protein A-coated ssDNA. But, more recently, it was noted t...
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Citations
... DSBs are repaired through two major repair pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ) in humans [14][15][16][17]. The molecular mechanism of DSB repair has been studied, and we now have good knowledge of the basic mechanisms of HR and NHEJ [18][19][20]. Both HR and NHEJ consist of multistep enzymatic processes containing many DNA repair factors. ...
Ionizing radiation (IR) and chemotherapy with DNA-damaging drugs such as cisplatin are vital cancer treatment options. These treatments induce double-strand breaks (DSBs) as cytotoxic DNA damage; thus, the DSB repair activity in each cancer cell significantly influences the efficacy of the treatments. Pancreatic cancers are known to be resistant to these treatments, and the overexpression of MUC1, a member of the glycoprotein mucins, is associated with IR- and chemo-resistance. Therefore, we investigated the impact of MUC1 on DSB repair. This report examined the effect of the overexpression of MUC1 on homologous recombination (HR) and non-homologous end-joining (NHEJ) using cell-based DSB repair assays. In addition, the therapeutic potential of NHEJ inhibitors including HDAC inhibitors was also studied using pancreatic cancer cell lines. The MUC1-overexpression enhances NHEJ, while partially suppressing HR. Also, MUC1-overexpressed cancer cell lines are preferentially killed by a DNA-PK inhibitor and HDAC1/2 inhibitors. Altogether, MUC1 induces metabolic changes that create an imbalance between NHEJ and HR activities, and this imbalance can be a target for selective killing by HDAC inhibitors. This is a novel mechanism of MUC1-mediated IR-resistance and will form the basis for targeting MUC1-overexpressed pancreatic cancer.
... In combination with the CRISPR/Cas9 system, "donor" plasmids with transgenes flanked by genomic homology regions are employed to mediate cellular homology-directed repair (HDR) pathways. 39 CRISPR/Cas9 induce double-strand breaks at target sites in the genome, activating cellular intrinsic DNA damage response and repair pathways. 40,41 In contrast to non-homology end-joining (NHEJ), in which broken ends of DNA are joined together to generate insertions and deletions (known as 'indels'), 42,43 donor-mediated HDR pathways faithfully reconstruct the genomic sequence, allowing precise integration of genes of interest in a locus-specific manner. ...
Self‐organizing brain organoids provide a promising tool for studying human development and disease. Here we created human forebrain organoids with stable and homogeneous expression of channelrhodopsin‐2 (ChR2) by generating AAVS1 safe harbor locus‐targeted, ChR2 knocked‐in human pluripotent stem cells (hPSCs), followed by the differentiation of these genetically engineered hPSCs into forebrain organoids. The resulting ChR2‐expressing human forebrain organoids showed homogeneous cellular expression of ChR2 throughout entire regions without any structural and functional perturbations and displayed consistent and robust neural activation upon light stimulation, allowing for the non‐virus mediated, spatiotemporal optogenetic control of neural activities. Furthermore, in the hybrid platform in which brain organoids are connected with spinal cord organoids and skeletal muscle spheroids, ChR2 knocked‐in forebrain organoids induced strong and consistent muscle contraction upon brain‐specific optogenetic stimulation. Our study thus provides a novel, non‐virus mediated, preclinical human organoid system for light‐inducible, consistent control of neural activities to study neural circuits and dynamics in normal and disease‐specific human brains as well as neural connections between brain and other peripheral tissues.
... These repair systems have varying levels of accuracy and efficiency. 49,50 HDR results in precise genome modification using a homologous repair template. Whereas, in contrast, NHEJ results in small random insertions and/or deletions (indels) at the cleavage site ( Figure 3). ...
The genome editing approach by clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is a revolutionary advancement in genetic engineering. Owing to its simple design and powerful genome-editing capability, it offers a promising strategy for the treatment of different infectious, metabolic, and genetic diseases. The crystal structure of Streptococcus pyogenes Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 Å resolution reveals a groove accommodating sgRNA:DNA heteroduplex within a bilobate architecture with target recognition (REC) and nuclease (NUC) domains. The presence of a PAM is significantly required for target recognition, R-loop formation, and strand scission. Recently, the spatiotemporal control of CRISPR/Cas9 genome editing has been considerably improved by genetic, chemical, and physical regulatory strategies. The use of genetic modifiers anti-CRISPR proteins, cell-specific promoters, and histone acetyl transferases has uplifted the application of CRISPR/Cas9 as a future-generation genome editing tool. In addition, interventions by chemical control, small-molecule activators, oligonucleotide conjugates and bioresponsive delivery carriers have improved its application in other areas of biological fields. Furthermore, the intermediation of physical control by using heat-, light-, magnetism-, and ultrasound-responsive elements attached to this molecular tool has revolutionized genome editing further. These strategies significantly reduce CRISPR/Cas9’s undesirable off-target effects. However, other undesirable effects still offer some challenges for comprehensive clinical translation using this genome-editing approach. In this review, we summarize recent advances in CRISPR/Cas9 structure, mechanistic action, and the role of small-molecule activators, inhibitors, promoters, and physical approaches. Finally, off-target measurement approaches, challenges, future prospects, and clinical applications are discussed.
... Most SSBs are repaired, the reparation process being carried out by the SSB repair system [14,21], while repairing DSBs is more complex. The two major systems of DDR to repair DSBs are the highly precise homologous recombination (HR) and non-homologous end-joining (NHEJ), which is more error-prone [17,22,23], since it ligates broken DNA ends together without a template [15][16][17]22,24,25]. Tumor cells are often HR-deficient and heavily rely on NHEJ for DSB repair, since impaired DDR and especially DSB repair results in genomic instability, which is a hallmark of cancer [19,21,[26][27][28][29]. ...
(1) Head and neck squamous cell carcinoma (HNSCC) is common, while treatment is difficult, and mortality is high. Kinase inhibitors are promising to enhance the effects of radiotherapy. We compared the effects of the PARP inhibitors talazoparib and niraparib and that of the DNA-PKcs inhibitor AZD7648, combined with ionizing radiation. (2) Seven HNSCC cell lines, including Cal33, CLS-354, Detroit 562, HSC4, RPMI2650 (HPV-negative), UD-SCC-2 and UM-SCC-47 (HPV-positive), and two healthy fibroblast cell lines, SBLF8 and SBLF9, were studied. Flow cytometry was used to analyze apoptosis and necrosis induction (AnnexinV/7AAD) and cell cycle distribution (Hoechst). Cell inactivation was studied by the colony-forming assay. (3) AZD7648 had the strongest effects, radiosensitizing all HNSCC cell lines, almost always in a supra-additive manner. Talazoparib and niraparib were effective in both HPV-positive cell lines but only consistently in one and two HPV-negative cell lines, respectively. Healthy fibroblasts were not affected by any combined treatment in apoptosis and necrosis induction or G2/M-phase arrest. AZD7648 alone was not toxic to healthy fibroblasts, while the combination with ionizing radiation reduced clonogenicity. (4) In conclusion, talazoparib, niraparib and, most potently, AZD7648 could improve radiation therapy in HNSCC. Healthy fibroblasts tolerated AZD7648 alone extremely well, but irradiation-induced effects might occur. Our results justify in vivo studies.
... The non-homologous end joining causes frameshift mutations, premature stop codons, and gene knockouts within the target zone by arbitrary insertions and deletions. While homology-directed repair produces precise changes to DNA utilizing an exogenous template, the procedure is restricted to active dividing cells, unlike post-mitotic cells such as photoreceptors and Müller glial cells [47,48]. Base editing was developed to overcome the limitations of the RNA-guided CRISPR-Cas nuclease system. ...
Pathogenic variants in the Crumbs homolog 1 (CRB1) gene lead to severe, childhood-onset retinal degeneration leading to blindness in early adulthood. There are no approved therapies, and traditional adeno-associated viral vector-based gene therapy approaches are challenged by the existence of multiple CRB1 isoforms. Here, we describe three CRB1 variants, including a novel, previously unreported variant that led to retinal degeneration. We offer a CRISPR-Cas-mediated DNA base editing strategy as a potential future therapeutic approach. This study is a retrospective case series. Clinical and genetic assessments were performed, including deep phenotyping by retinal imaging. In silico analyses were used to predict the pathogenicity of the novel variant and to determine whether the variants are amenable to DNA base editing strategies. Case 1 was a 24-year-old male with cone–rod dystrophy and retinal thickening typical of CRB1 retinopathy. He had a relatively preserved central outer retinal structure and a best corrected visual acuity (BCVA) of 60 ETDRS letters in both eyes. Genetic testing revealed compound heterozygous variants in exon 9: c.2843G>A, p.(Cys948Tyr) and a novel variant, c.2833G>A, p.(Gly945Arg), which was predicted to likely be pathogenic by an in silico analysis. Cases 2 and 3 were two brothers, aged 20 and 24, who presented with severe cone–rod dystrophy and a significant disruption of the outer nuclear layers. The BCVA was reduced to hand movements in both eyes in Case 2 and to 42 ETDRS letters in both eyes in Case 3. Case 2 was also affected with marked cystoid macular lesions, which are common in CRB1 retinopathy, but responded well to treatment with oral acetazolamide. Genetic testing revealed two c.2234C>T, p.(Thr745Met) variants in both brothers. As G-to-A and C-to-T variants, all three variants are amenable to adenine base editors (ABEs) targeting the forward strand in the Case 1 variants and the reverse strand in Cases 2 and 3. Available PAM sites were detected for KKH-nSaCas9-ABE8e for the c.2843G>A variant, nSaCas9-ABE8e and KKH-nSaCas9-ABE8e for the c.2833G>A variant, and nSpCas9-ABE8e for the c.2234C>T variant. In this case series, we report three pathogenic CRB1 variants, including a novel c.2833G>A variant associated with early-onset cone–rod dystrophy. We highlight the severity and rapid progression of the disease and offer ABEs as a potential future therapeutic approach for this devastating blinding condition.
... Double-strand breaks (DSBs), gaps, and interstrand crosslinks (ICLs) in DNA disrupt normal cellular functions and, if left unrepaired, can lead to cell death. HR is a highly conserved pathway that repairs such damages by utilizing an intact DNA template from the sister chromatid or homologous chromosome when available (1,2). DNA resection, homology search, DNA strand exchange, and new DNA synthesis encompass the central steps of HR, while subsequent steps determine the amount and configuration of new genetic information incorporated into the repaired DNA. ...
Homologous recombination (HR) is a high-fidelity repair mechanism for double-strand breaks. Rad51 is the key enzyme that forms filaments on single-stranded DNA (ssDNA) to catalyze homology search and DNA strand exchange in recombinational DNA repair. In this study, we employed single-particle cryo-electron microscopy (cryo-EM) to ascertain the density map of the budding yeast Rad51-ssDNA filament bound to ADP-AlF3, achieving a resolution of 2.35 Angstrom without imposing helical symmetry. The model assigned 6 Rad51 protomers, 24 nt of DNA, and 6 bound ADP-AlF3. It shows 6-fold symmetry implying monomeric building blocks, unlike the structure of the Rad51-I345T mutant filament with three-fold symmetry implying dimeric building blocks, for which the structural comparisons provide a satisfying mechanistic explanation. This image analysis enables comprehensive comparisons of individual Rad51 protomers within the filament and reveals local conformational movements of amino acid side chains. Notably, Arg293 in Loop1 adopts multiple conformations to facilitate Leu296 and Val331 in separating and twisting the DNA triplets. We also analyzed the predicted structures of yeast Rad51-K342E and two tumor-derived human RAD51 variants, RAD51-Q268P and RAD51-Q272L, using the Rad51-ssDNA structure from this study as a reference.
... In brief, PARP1 binds to the ends of DSBs as a competitor of Ku that is known to play a similar role in the NHEJ pathway [16] and activates the end resection [17], thus channeling DNA repair to the MMEJ pathway. RPA that contains three subunits-RPA1, RPA2, and RPA3binds resected single-stranded DNA (ssDNA) and triggers homologous recombination (HR) [18,19]. RPA also prevents ssDNA annealing thus further blocking MMEJ repair [20]. ...
Background
CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks.
Results
Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells.
Conclusions
Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.
... Strand invasion is followed by repair synthesis. In SDSA, the newly synthesized strand dissociates, allowing the DNA ends to anneal, ligate, and create a noncrossover product that may include gene conversion [6,[12][13][14]. DSBR involves second end-capture to create a double Holliday junction (dHJ). ...
... DSBR involves second end-capture to create a double Holliday junction (dHJ). Depending on how the dHJ is resolved by endonucleases, the product can be either a crossover or a noncrossover [8,14] that may also include gene conversion. ...
Accurate repair of DNA double-strand breaks (DSBs) is essential for the maintenance of genome integrity, as failure to repair DSBs can result in cell death. The cell has evolved two main mechanisms for DSB repair: non-homologous end-joining (NHEJ) and homology-directed repair (HDR), which includes single-strand annealing (SSA) and homologous recombination (HR). While certain factors like age and state of the chromatin are known to influence DSB repair pathway choice, the roles of developmental stage, tissue type, and sex have yet to be elucidated in multicellular organisms. To examine the influence of these factors, DSB repair in various embryonic developmental stages, larva, and adult tissues in Drosophila melanogaster was analyzed through molecular analysis of the DR- white assay using Tracking across Indels by DEcomposition (TIDE). The proportion of HR repair was highest in tissues that maintain the canonical (G1/S/G2/M) cell cycle and suppressed in both terminally differentiated and polyploid tissues. To determine the impact of sex on repair pathway choice, repair in different tissues in both males and females was analyzed. When molecularly examining tissues containing mostly somatic cells, males and females demonstrated similar proportions of HR and NHEJ. However, when DSB repair was analyzed in male and female premeiotic germline cells utilizing phenotypic analysis of the DR- white assay, there was a significant decrease in HR in females compared to males. This study describes the impact of development, tissue-specific cycling profile, and, in some cases, sex on DSB repair outcomes, underscoring the complexity of repair in multicellular organisms.
... The formed complex BRCA1-PALB2-BRCA2 finally removes RPA and promotes the assembly of the RAD51 recombinase nucleoprotein filament. The RAD51 recombinase nucleoprotein filament is the actual effector of the invasion of ssDNA into the undamaged sister chromatid to find the homologous sequence for DNA polymerase to use as a template for DNA synthesis [17,33]. ...
More than 20% of metastatic prostate cancer carries genomic defects involving DNA damage repair pathways, mainly in homologous recombination repair-related genes. The recent approval of olaparib has paved the way to precision medicine for the treatment of metastatic prostate cancer with PARP inhibitors in this subset of patients, especially in the case of BRCA1 or BRCA2 pathogenic/likely pathogenic variants. In face of this new therapeutic opportunity, many issues remain unsolved. This narrative review aims to describe the relationship between homologous recombination repair deficiency and prostate cancer, the techniques used to determine homologous recombination repair status in prostate cancer, the crosstalk between homologous recombination repair and the androgen receptor pathway, the current evidence on PARP inhibitors activity in metastatic prostate cancer also in homologous recombination repair-proficient tumors, as well as emerging mechanisms of resistance to PARP inhibitors. The possibility of combination therapies including a PARP inhibitor is an attractive option, and more robust data are awaited from ongoing phase II and phase III trials outlined in this manuscript.
... However, TLS has increased mutation rates due to loose active sites and lack of proofreading activity in TLS polymerases (11). In contrast, the error-free DNA lesion bypass, mediated by homologous recombination (HR), does not cause additional mutations (12). The highly conserved Rad51 recombinase protein and its paralogs play essential roles in error-free lesion bypass. ...
... The highly conserved Rad51 recombinase protein and its paralogs play essential roles in error-free lesion bypass. Rad51 binds single-stranded DNA (ssDNA) to form stable filaments for HR actions (5,12). The hallmark of the error-free pathway is to use homologous sister chromatids as the templates to ensure faithful replication (5,12). ...
... Rad51 binds single-stranded DNA (ssDNA) to form stable filaments for HR actions (5,12). The hallmark of the error-free pathway is to use homologous sister chromatids as the templates to ensure faithful replication (5,12). Rad51 filaments mediate essential steps associated with template switching to ensure genomic integrity. ...
Rad51 filaments are Rad51-coated single-stranded DNA and essential intermediates in homologous recombination (HR) and the HR-associated DNA damage response. The yeast Shu complex (Shu) is a conserved regulator of HR, working through its modulation of Rad51 filaments. However, the biochemical properties of Shu remain unclear, which hinders molecular insight into Shu’s role in HR and the DNA damage response. In this work, we biochemically characterized Shu and analyzed its molecular actions on single-stranded DNA and Rad51 filaments. First, we revealed that Shu preferentially binds DNA with ssDNA components and ssDNA/double-stranded DNA junctions. Then, we identified and validated, through site-specific mutagenesis, that Shu is an ATPase and hydrolyzes ATP in a DNA-dependent manner. Furthermore, we showed that Shu interacts with ssDNA and Rad51 filaments at the 5’ end preferentially, altering the conformations of ssDNA and the filaments. The alterations depend on the ATP hydrolysis of Shu, suggesting that the ATPase activity of Shu is important in regulating its functions in HR. The preference of Shu for acting on the 5’ end of Rad51 filaments aligns with the observation that Shu promotes lesion bypass at the lagging strand of a replication fork. Our work on Shu, a prototype modulator of Rad51 filaments in eukaryotes, provides a general molecular mechanism for Rad51-mediated error-free DNA lesion bypass.
