Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms

DNA double stranded breaks (DSBs) are one of the most deleterious types of DNA lesions. The main pathways responsible for repairing these breaks in eukaryotic cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). However, a third group of still poorly characterized DSB repair pathways, collectively termed microhomology-mediated end-joining (MMEJ), relies on short homologies for the end-joining process. Here, we constructed GFP reporter assays to characterize and distinguish MMEJ variant pathways, namely the simple MMEJ and the DNA synthesis-dependent (SD)-MMEJ mechanisms. Transfection of these assay vectors in Chinese hamster ovary (CHO) cells and characterization of the repaired DNA sequences indicated that while simple MMEJ is able to mediate relatively efficient DSB repair if longer microhomologies are present, the majority of DSBs were repaired using the highly error-prone SD-MMEJ pathway. To validate the involvement of DNA synthesis in the repair process, siRNA knock-down of different genes proposed to play a role in MMEJ were performed, revealing that the knock-down of DNA polymerase θ inhibited DNA end resection and repair through simple MMEJ, thus favoring the other repair pathway. Overall, we conclude that this approach provides a convenient assay to study MMEJ-related DNA repair pathways.     

Capture of extranuclear DNA at fission yeast double-strand breaks

Proper repair of DNA double-strand breaks (DSBs) is necessary for the maintenance of genomic integrity. Here, a new simple assay was used to study extrachromosomal DSB repair in Schizosaccharomyces pombe. Strikingly, DSB repair was associated with the capture of fission yeast mitochondrial DNA (mtDNA) at high frequency. Capture of mtDNA fragments required the Lig4p/Pku70p nonhomologous end-joining (NHEJ) machinery and its frequency was highly increased in fission yeast cells grown to stationary phase. The fission yeast Mre11 complex Rad32p/Rad50p/Nbs1p was also required for efficient capture of mtDNA at DSBs, supporting a role for the complex in promoting intermolecular ligation. Competition assays further revealed that microsatellite DNA from higher eukaryotes was preferentially captured at yeast DSBs. Finally, cotransformation experiments indicated that, in NHEJ-deficient cells, capture of extranuclear DNA at DSBs was observed if homologies--as short as 8 bp--were present between DNA substrate and DSB ends. Hence, whether driven by NHEJ, microhomology-mediated end-joining, or homologous recombination, DNA capture associated with DSB repair is a mutagenic process threatening genomic stability.     

Microhomology-mediated deletion and gene conversion in African trypanosomes

Antigenic variation in African trypanosomes is induced by DNA double-strand breaks (DSBs). In these protozoan parasites, DSB repair (DSBR) is dominated by homologous recombination (HR) and microhomology-mediated end joining (MMEJ), while non-homologous end joining (NHEJ) has not been reported. To facilitate the analysis of chromosomal end-joining, we established a system whereby inter-allelic repair by HR is lethal due to loss of an essential gene. Analysis of intrachromosomal end joining in individual DSBR survivors exclusively revealed MMEJ-based deletions but no NHEJ. A survey of microhomologies typically revealed sequences of between 5 and 20 bp in length with several mismatches tolerated in longer stretches. Mean deletions were of 54 bp on the side closest to the break and 284 bp in total. Break proximity, microhomology length and GC-content all favored repair and the pattern of MMEJ described above was similar at several different loci across the genome. We also identified interchromosomal gene conversion involving HR and MMEJ at different ends of a duplicated sequence. While MMEJ-based deletions were RAD51-independent, one-sided MMEJ was RAD51 dependent. Thus, we describe the features of MMEJ in Trypanosoma brucei, which is analogous to micro single-strand annealing; and RAD51 dependent, one-sided MMEJ. We discuss the contribution of MMEJ pathways to genome evolution, subtelomere recombination and antigenic variation.     

The Mre11/Rad50/Nbs1 complex functions in resection-based DNA end joining in Xenopus laevis

The repair of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. In higher eukaryotes, DNA DSBs are predominantly repaired by non-homologous end joining (NHEJ), but DNA ends can also be joined by an alternative error-prone mechanism termed microhomology-mediated end joining (MMEJ). In MMEJ, the repair of DNA breaks is mediated by annealing at regions of microhomology and is always associated with deletions at the break site. In budding yeast, the Mre11/Rad5/Xrs2 complex has been demonstrated to play a role in both classical NHEJ and MMEJ, but the involvement of the analogous MRE11/RAD50/NBS1 (MRN) complex in end joining in higher eukaryotes is less certain. Here we demonstrate that in Xenopus laevis egg extracts, the MRN complex is not required for classical DNA-PK-dependent NHEJ. However, the XMRN complex is necessary for resection-based end joining of mismatched DNA ends. This XMRN-dependent end joining process is independent of the core NHEJ components Ku70 and DNA-PK, occurs with delayed kinetics relative to classical NHEJ and brings about repair at sites of microhomology. These data indicate a role for the X. laevis MRN complex in MMEJ.     

Microhomology-mediated end joining in fission yeast is repressed by pku70 and relies on genes involved in homologous recombination

Two DNA repair pathways are known to mediate DNA double-strand-break (DSB) repair: homologous recombination (HR) and nonhomologous end joining (NHEJ). In addition, a nonconservative backup pathway showing extensive nucleotide loss and relying on microhomologies at repair junctions was identified in NHEJ-deficient cells from a variety of organisms and found to be involved in chromosomal translocations. Here, an extrachromosomal assay was used to characterize this microhomology-mediated end-joining (MMEJ) mechanism in fission yeast. MMEJ was found to require at least five homologous nucleotides and its efficiency was decreased by the presence of nonhomologous nucleotides either within the overlapping sequences or at DSB ends. Exo1 exonuclease and Rad22, a Rad52 homolog, were required for repair, suggesting that MMEJ is related to the single-strand-annealing (SSA) pathway of HR. In addition, MMEJ-dependent repair of DSBs with discontinuous microhomologies was strictly dependent on Pol4, a PolX DNA polymerase. Although not strictly required, Msh2 and Pms1 mismatch repair proteins affected the pattern of MMEJ repair. Strikingly, Pku70 inhibited MMEJ and increased the minimal homology length required for efficient MMEJ. Overall, this study strongly suggests that MMEJ does not define a distinct DSB repair mechanism but reflects "micro-SSA."     

PARP-3 and APLF function together to accelerate nonhomologous end-joining

PARP-3 is a member of the ADP-ribosyl transferase superfamily of unknown function. We show that PARP-3 is stimulated by DNA double-strand breaks (DSBs) in vitro and functions in the same pathway as the poly (ADP-ribose)-binding protein APLF to accelerate chromosomal DNA DSB repair. We implicate PARP-3 in the accumulation of APLF at DSBs and demonstrate that APLF promotes the retention of XRCC4/DNA ligase IV complex in chromatin, suggesting that PARP-3 and APLF accelerate DNA ligation during nonhomologous end-joining (NHEJ). Consistent with this, we show that class switch recombination in Aplf(-/-) B cells is biased toward microhomology-mediated end-joining, a pathway that operates in the absence of XRCC4/DNA ligase IV, and that the requirement for PARP-3 and APLF for NHEJ is circumvented by overexpression of XRCC4/DNA ligase IV. These data identify molecular roles for PARP-3 and APLF in chromosomal DNA double-strand break repair reactions.     

Non-homologous DNA end joining

DNA double strand breaks (DSB) are the most serious form of DNA damage. Repair of DSBs is important to prevent chromosomal fragmentation, translocations and deletions. Non-homologous end joining (NHEJ) is one of three major pathways for the repair of DSBs in human cells. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. NHEJ is typically imprecise, a characteristic that is useful for immune diversification in lymphocytes in V(D)J recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku proteins, XRCC4, DNA ligase IV, and Artemis. This review focuses on the mechanisms an dregulation of DSB repair by NHEJ in mammalian cells.     

Targeting abnormal DNA repair in therapy-resistant breast cancers

Although hereditary breast cancers have defects in the DNA damage response that result in genomic instability, DNA repair abnormalities in sporadic breast cancers have not been extensively characterized. Recently, we showed that, relative to nontumorigenic breast epithelial MCF10A cells, estrogen receptor-positive (ER+) MCF7 breast cancer cells and progesterone receptor-positive (PR+) MCF7 breast cancer cells have reduced steady-state levels of DNA ligase IV, a component of the major DNA-protein kinase (PK)-dependent nonhomologous end joining (NHEJ) pathway, whereas the steady-state level of DNA ligase IIIα, a component of the highly error-prone alternative NHEJ (ALT NHEJ) pathway, is increased. Here, we show that tamoxifen- and aromatase-resistant derivatives of MCF7 cells and ER(-)/PR(-) cells have even higher steady-state levels of DNA ligase IIIα and increased levels of PARP1, another ALT NHEJ component. This results in increased dependence upon microhomology-mediated ALT NHEJ to repair DNA double-strand breaks (DSB) and the accumulation of chromosomal deletions. Notably, therapy-resistant derivatives of MCF7 cells and ER(-)/PR(-) cells exhibited significantly increased sensitivity to a combination of PARP and DNA ligase III inhibitors that increased the number of DSBs. Biopsies from ER(-)/PR(-) tumors had elevated levels of ALT NHEJ and reduced levels of DNA-PK-dependent NHEJ factors. Thus, our results show that ALT NHEJ is a novel therapeutic target in breast cancers that are resistant to frontline therapies and suggest that changes in NHEJ protein levels may serve as biomarkers to identify tumors that are candidates for this therapeutic approach.     

Involvement of Ku80 in microhomology-mediated end joining for DNA double-strand breaks in vivo

Mammalian cells have an activity of mutagenic repair for DNA double-strand breaks (DSBs), microhomology-mediated end joining (MMEJ), in which DNA ends are joined via microhomologous sequences flanking the breakpoint. MMEJ has been indicated to be undertaken without Ku proteins, which are essential factors for non-homologous end joining (NHEJ). On the other hand, recent studies with cell-free (in vitro) systems indicated the involvement of Ku proteins in MMEJ, suggesting that MMEJ could be also undertaken by a Ku-dependent pathway. To clarify whether Ku proteins are essential in MMEJ in vivo, linearized plasmid DNAs with microhomologous sequences of 10bp at both ends were introduced as repair substrates into Ku80-proficient and Ku80-deficient CHO cells, and were subjected to MMEJ and NHEJ. Activities of MMEJ and NHEJ, respectively, of the cells were evaluated by mathematical modeling for the increase in fluorescence of GFP proteins produced from repaired products. The Ku80 deficiency caused approximately 75% reduction of the MMEJ activity in CHO cells, while it caused is > or =90% reduction of the NHEJ activity. Therefore, it was indicated that there is a Ku-dependent pathway for MMEJ; however, MMEJ is less dependent on Ku80 protein than NHEJ. The fraction of MMEJ products increased in proportion to the increase in the amounts of substrates. The results suggest that the increase in DSBs makes the cell more predominant for MMEJ. MMEJ might function as a salvage pathway for DSBs that cannot be repaired by NHEJ.     

Nuclear mitochondrial pseudogenes

As has been demonstrated recently, the transfer of genetic material from mitochondria to the nucleus and its integration into the nuclear genome is a continuous and dynamic process. Fragments of mitochondrial DNA (mtDNA) are incorporated in the nuclear genome as noncoding sequences, which are called nuclear mitochondrial pseudogenes (NUMT pseudogenes or NUMT inserts). In various eukaryotes, NUMT pseudogenes are distributed through different chromosomes to form a “library” of mtDNA fragments, providing important information on genome evolution. The escape of mtDNA from mitochondria is mostly associated with mitochondrial damage and mitophagy. Fragments of mtDNA may be integrated into nuclear DNA (nDNA) during repair of double-strand breaks (DSBs), which are caused by endogenous or exogenous agents. DSB repair of nDNA with a capture of mtDNA fragments may occur via nonhomologous end joining or a similar mechanism that involves microhomologous terminal sequences. An analysis of the available data makes it possible to suppose that the NUMT pseudogene formation rate depends on the DSB rate in nDNA, the activity of the repair systems, and the number of mtDNA fragments leaving organelles and migrating into the nucleus. Such situations are likely after exposure to damaging agents, first and foremost, ionizing radiation. Not only do new NUMT pseudogenes change the genome structure in the regions of their integration, but they may also have a significant impact on the actualization of genetic information. The de novo integration of NUMT pseudogenes in the nuclear genome may play a role in various pathologies and aging. NUMT pseudogenes may cause errors in PCR-based analyses of free mtDNA as a component of total cell DNA because of their coamplification.     

Time-dependent predominance of nonhomologous DNA end-joining pathways during embryonic development in mice

Repair of DNA double-strand breaks (DSBs) is crucial for maintaining genomic integrity during the successful development of a fertilized egg into a whole organism. To date, the mechanism of DSB repair in postimplantation embryos has been largely unknown. In the present study, using a cell-free repair system derived from the different embryonic stages of mice, we find that canonical nonhomologous end joining (NHEJ), one of the major DSB repair pathways in mammals, is predominant at 14.5 day of embryonic development. Interestingly, all four types of DSBs tested were repaired by ligase IV/XRCC4 and Ku-dependent classical NHEJ. Characterization of end-joined junctions and expression studies further showed evidences for canonical NHEJ. Strikingly, in contrast to the above, we observed noncanonical end joining accompanied by DSB resection, dependent on microhomology and ligase III in 18.5-day embryos. Interestingly, we observed an elevated expression of CtIP, MRE11, and NBS1 at this stage, suggesting that it could act as a switch between classical end joining and microhomology-mediated end joining at later stages of embryonic development. Thus, our results establish for the first time the existence of both canonical and alternative NHEJ pathways during the postimplantation stages of mammalian embryonic development.     

ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining

The human disorder ataxia telangiectasia (AT), which is characterized by genetic instability and neurodegeneration, results from mutation of the ataxia telangiectasia mutated (ATM) kinase. The loss of ATM leads to cell-cycle checkpoint deficiencies and other DNA damage signaling defects that do not fully explain all pathologies associated with A-T including neuronal loss. In addressing this enigma, we find here that ATM suppresses DNA double-strand break (DSB) repair by microhomology-mediated end joining (MMEJ). We show that ATM repression of DNA end-degradation is dependent on its kinase activities and that Mre11 is the major nuclease behind increased DNA end-degradation and MMEJ repair in A-T. Assessment of MMEJ by an in vivo reporter assay system reveals decreased levels of MMEJ repair in Mre11-knockdown cells and in cells treated with Mre11-nuclease inhibitor mirin. Structure-based modeling of Mre11 dimer engaging DNA ends suggests the 5' ends of a bridged DSB are juxtaposed such that DNA unwinding and 3'-5' exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5' termini and exonucleolytic degradation of the 3' ends in MMEJ. Together our results provide an integrated understanding of ATM and Mre11 in MMEJ: ATM has a critical regulatory function in controlling DNA end-stability and error-prone DSB repair and Mre11 nuclease plays a major role in initiating MMEJ in mammalian cells. These functions of ATM and Mre11 could be particularly important in neuronal cells, which are post-mitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs.     

New Insights into NHEJ Repair Processes in Prokaryotes

In eukaryotic cells, the repair of DNA double strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway is critical for genome stability. Until recently it was assumed that this DSB repair pathway was restricted to the eukarya. However, a functionally homologous prokaryotic NHEJ repair apparatus has now been identified and characterised. In contrast to the complex eukaryotic system, bacterial NHEJ appears to require only two proteins, Ku and a multifunctional DNA ligase, which form a two-component repair complex at the termini of DSBs. Together, these DNA repair factors possess all of the break-recognition, end-processing and ligation activities required to facilitate the complex task of DSB repair, both in vitro and in vivo. Our recent findings lay the foundation for understanding the molecular mechanisms that co-ordinate the processing and joining of DSBs by NHEJ in bacteria and also provides a conceptual framework for delineating the end-processing reactions in eukaryotes.     

ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining

The human disorder ataxia telangiectasia (AT), which is characterized by genetic instability and neurodegeneration, results from mutation of the ataxia telangiectasia mutated (ATM) kinase. The loss of ATM leads to cell cycle checkpoint deficiencies and other DNA damage signaling defects that do not fully explain all pathologies associated with A-T including neuronal loss. In addressing this enigma, we find here that ATM suppresses DNA double-strand break (DSB) repair by microhomology-mediated end joining (MMEJ). We show that ATM repression of DNA end-degradation is dependent on its kinase activities and that Mre11 is the major nuclease behind increased DNA end-degradation and MMEJ repair in A-T. Assessment of MMEJ by an in vivo reporter assay system reveals decreased levels of MMEJ repair in Mre11-knockdown cells and in cells treated with Mre11-nuclease inhibitor mirin. Structure-based modeling of Mre11 dimer engaging DNA ends suggests the 5′ ends of a bridged DSB are juxtaposed such that DNA unwinding and 3′–5′ exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5′ termini and exonucleolytic degradation of the 3′ ends in MMEJ. Together our results provide an integrated understanding of ATM and Mre11 in MMEJ: ATM has a critical regulatory function in controlling DNA end-stability and error-prone DSB repair and Mre11 nuclease plays a major role in initiating MMEJ in mammalian cells. These functions of ATM and Mre11 could be particularly important in neuronal cells, which are post-mitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs.Key words: ATM, Mre11, MRN complex, DNA degradation, double-strand break repair, microhomology-mediated end joining, PI-3-kinase-like kinases     

Microhomology-mediated DNA strand annealing and elongation by human DNA polymerases λ and β on normal and repetitive DNA sequences

'Classical' non-homologous end joining (NHEJ), dependent on the Ku70/80 and the DNA ligase IV/XRCC4 complexes, is essential for the repair of DNA double-strand breaks. Eukaryotic cells possess also an alternative microhomology-mediated end-joining (MMEJ) mechanism, which is independent from Ku and DNA ligase 4/XRCC4. The components of the MMEJ machinery are still largely unknown. Family X DNA polymerases (pols) are involved in the classical NHEJ pathway. We have compared in this work, the ability of human family X DNA pols β, λ and μ, to promote the MMEJ of different model templates with terminal microhomology regions. Our results reveal that DNA pol λ and DNA ligase I are sufficient to promote efficient MMEJ repair of broken DNA ends in vitro, and this in the absence of auxiliary factors. However, DNA pol β, not λ, was more efficient in promoting MMEJ of DNA ends containing the (CAG)n triplet repeat sequence of the human Huntingtin gene, leading to triplet expansion. The checkpoint complex Rad9/Hus1/Rad1 promoted end joining by DNA pol λ on non-repetitive sequences, while it limited triplet expansion by DNA pol β. We propose a possible novel role of DNA pol β in MMEJ, promoting (CAG)n triplet repeats instability.     

组蛋白去乙酰化酶抑制剂对DNA双链断裂修复路径的作用

DNA双链断裂(double strand breaks, DSBs)对细胞生存是致命的.细胞内非同源末端连接(NHEJ)、重组修复(HDR)、单链退火修复(SSA)和微同源序列末端连接(MMEJ)等通路可竞争性修复DNA双链断裂损伤.在肿瘤细胞DNA中制造难以修复的基因损伤,诱导肿瘤细胞周期中止、坏死和凋亡是临床放、化疗的主要策略.组蛋白去乙酰化酶（histone deacetylase）作为抗肿瘤治疗的新靶标,其抑制剂(histonedeacetylase inhibitors, HDACi)可显著降低肿瘤细胞DSBs修复能力,增强肿瘤细胞的放、化疗敏感性.研究显示,HDACi抑制了肿瘤细胞中具有正确修复倾向的HDR和经典NHEJ通路,具有错误修复倾向的SSA和MMEJ路径也可能牵涉其中.目前,HDACi作用于DSBs修复通路的分子机制已取得较大进展,但仍有许多问题有待阐明.     

Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae

Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.     

Role of budding yeast Rad18 in repair of HO-induced double-strand breaks

The Rad6-Rad18 complex mono-ubiquitinates proliferating cell nuclear antigen (PCNA) at the lysine 164 residue after DNA damage and promotes DNA polymerase eta (Poleta)- and Polzeta/Rev1-dependent DNA synthesis. Double-strand breaks (DSBs) of DNA can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ), both of which require new DNA synthesis. HO endonuclease introduces DSBs into specific DNA sequences. We have shown that Polzeta and Rev1 localize to HO-induced DSBs in a Mec1-dependent manner and promote Ku-dependent DSB repair. However, Polzeta and Rev1 localize to DSBs independently of PCNA ubiquitination. Here we provide evidence indicating that Rad18-mediated PCNA ubiquitination stimulates DNA synthesis by Polzeta and Rev1 in repair of HO-induced DSBs. Ubiquitination defective PCNA mutation or rad18Delta mutation confers the same DSB repair defect as rev1Delta mutation. Consistent with a role in DSB repair, Rad18 localizes to HO-induced DSBs in a Rad6-dependent manner. Unlike Polzeta or Rev1, Poleta is dispensable for repair of HO-induced DSBs. Ku and DNA ligase IV constitute a central NHEJ pathway. We also show that Polzeta and Rev1 act in the same pathway as DNA ligase IV, suggesting that Polzeta and Rev1 are involved in DNA synthesis during NHEJ. Our results suggest that Polzeta-Rev1 accumulates at regions near DSBs independently of PCNA ubiquitination and then interacts with ubiquitinated PCNA to facilitate DNA synthesis.     

Recruitment and dissociation of nonhomologous end joining proteins at a DNA double-strand break in Saccharomyces cerevisiae

Nonhomologous end joining (NHEJ) is an important DNA double-strand-break (DSB) repair pathway that requires three protein complexes in Saccharomyces cerevisiae: the Ku heterodimer (Yku70-Yku80), MRX (Mre11-Rad50-Xrs2), and DNA ligase IV (Dnl4-Lif1), as well as the ligase-associated protein Nej1. Here we use chromatin immunoprecipitation from yeast to dissect the recruitment and release of these protein complexes at HO-endonuclease-induced DSBs undergoing productive NHEJ. Results revealed that Ku and MRX assembled at a DSB independently and rapidly after DSB formation. Ligase IV appeared at the DSB later than Ku and MRX and in a strongly Ku-dependent manner. Ligase binding was extensive but slightly delayed in rad50 yeast. Ligase IV binding occurred independently of Nej1, but instead promoted loading of Nej1. Interestingly, dissociation of Ku and ligase from unrepaired DSBs depended on the presence of an intact MRX complex and ATP binding by Rad50, suggesting a possible role of MRX in terminating a NHEJ repair phase. This activity correlated with extended DSB resection, but limited degradation of DSB ends occurred even in MRX mutants with persistently bound Ku. These findings reveal the in vivo assembly of the NHEJ repair complex and shed light on the mechanisms controlling DSB repair pathway utilization.     

Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis

DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp) deletions occurred at a minority (25-45%) of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.