Healing the wounds inflicted by sleeping beauty transposition by double-strand break repair in mammalian somatic cells

The Sleeping Beauty (SB) element is a useful tool to probe transposon-host interactions in vertebrates. We investigated requirements of DNA repair factors for SB transposition in mammalian cells. Factors of nonhomologous end joining (NHEJ), including Ku, DNA-PKcs, and Xrcc4 as well as Xrcc3/Rad51C, a complex that functions during homologous recombination, are required for efficient transposition. NHEJ plays a dominant role in repair of transposon excision sites in somatic cells. Artemis is dispensable for transposition, consistent with the lack of a hairpin structure at excision sites. Ku physically interacts with the SB transposase. DNA-PKcs is a limiting factor for transposition and, in addition to repair, has a function in transposition that is independent from its kinase activity. ATM is involved in excision site repair and affects transposition rates. The overlapping but distinct roles of repair factors in transposition and in V(D)J recombination might influence the outcomes of these mechanistically similar processes.     

Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair

Background  

Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.     

Comparative analysis of non-random DNA repair following Ac transposon excision in maize and Arabidopsis

Ac/Ds transposable elements often leave short DNA rearrangements, or 'footprints,' at the sites where they excise. Previous studies at the maize waxy ( wx ) gene suggest that the DNA repair that forms transposon footprints is not random. Each excision site consistently displays a different, predominant repair product suggesting flanking DNA may influence footprint formation. We have expanded these studies to show that predominant end-joining products also form in association with Ac/Ds excision in Arabidopsis and that chromosomal location of the Ac -containing construct does not appear to influence this repair. The predominant repair product is identical in both maize and Arabidopsis for Ac elements with the same adjacent DNA sequences. However, a broader range of minor footprint types is observed in Arabidopsis , including footprints that are rare in maize, suggesting potential differences in the host proteins involved in either transposition, repair or both. The data also suggest that the sequences influencing footprint formation are within 39 bp 5' and 18 bp 3' of the transposon. These studies demonstrate that transgenic Ac/Ds -containing plants will be useful tools in dissecting plant DNA repair processes.     

The Mre11/Rad50/Xrs2 complex and non-homologous end-joining of incompatible ends in S. cerevisiae

In Saccharomyces cerevisiae, the Mre11/Rad50/Xrs2 (MRX) complex plays important roles in both homologous and non-homologous pathways of DNA repair. In this study, we investigated the role of the MRX complex and its enzymatic functions in non-homologous repair of DNA ends containing incompatible end structures. Using a plasmid transformation assay, we found that mre11 and rad50 null strains are extremely deficient in joining of incompatible DNA ends. Expression of the nuclease-deficient Mre11 mutant H125N fully complemented the mre11 strain for joining of mismatched ends in the absence of homology, while a mutant of Rad50 deficient in ATP-dependent activities exhibited levels of end-joining similar to a rad50 deletion strain. Although the majority of non-homologous end-joining (NHEJ) products isolated did not contain microhomologies, introduction of an 8bp microhomology at mismatched ends resulted in microhomology-mediated joining in all of the products recovered, demonstrating that a microhomology exerts a dominant effect on processing events that occur during NHEJ. Nuclease-deficient Mre11p was less efficient in promoting microhomology-mediated end-joining in comparison to its ability to stimulate non-microhomology-mediated events, suggesting that Mre11p influences, but is not essential for, microhomology-mediated repair. When the linearized DNA was transformed in the presence of an intact homologous plasmid to facilitate gap repair, there was no decrease in NHEJ products obtained, suggesting that NHEJ and homologous repair do not compete for DNA ends in vivo. These results suggest that the MRX complex is essential for joining of incompatible ends by NHEJ, and the ATP-dependent activities of Rad50 are critical for this process.     

Transposon excision from an atypical site: a mechanism of evolution of novel transposable elements

The role of transposable elements in sculpting the genome is well appreciated but remains poorly understood. Some organisms, such as humans, do not have active transposons; however, transposable elements were presumably active in their ancestral genomes. Of specific interest is whether the DNA surrounding the sites of transposon excision become recombinogenic, thus bringing about homologous recombination. Previous studies in maize and Drosophila have provided conflicting evidence on whether transposon excision is correlated with homologous recombination. Here we take advantage of an atypical Dissociation (Ds) element, a maize transposon that can be mobilized by the Ac transposase gene in Arabidopsis thaliana, to address questions on the mechanism of Ds excision. This atypical Ds element contains an adjacent 598 base pairs (bp) inverted repeat; the element was allowed to excise by the introduction of an unlinked Ac transposase source through mating. Footprints at the excision site suggest a micro-homology mediated non-homologous end joining reminiscent of V(D)J recombination involving the formation of intra-helix 3' to 5' trans-esterification as an intermediate, a mechanism consistent with previous observations in maize, Antirrhinum and in certain insects. The proposed mechanism suggests that the broken chromosome at the excision site should not allow recombinational interaction with the homologous chromosome, and that the linked inverted repeat should also be mobilizable. To test the first prediction, we measured recombination of flanking chromosomal arms selected for the excision of Ds. In congruence with the model, Ds excision did not influence crossover recombination. Furthermore, evidence for correlated movement of the adjacent inverted repeat sequence is presented; its origin and movement suggest a novel mechanism for the evolution of repeated elements. Taken together these results suggest that the movement of transposable elements themselves may not directly influence linkage. Possibility remains, however, for novel repeated DNA sequences produced as a consequence of transposon movement to influence crossover in subsequent generations.     

Ac／Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(Ac TPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ae×Ds的杂交组合354个。检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性。结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4％。检测到T-DNA可插入到编码蛋白的基因中。在Ac×Ds的F2代中,Ds因子的转座频率为22.7％。对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制——转座和小完全切离等现象。获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录。探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略。     

Ac/Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(AcTPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ac×Ds的杂交组合354个.检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性.结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4%.检测到T-DNA可插入到编码蛋白的基因中.在Ac×Ds的F2代中,Ds因子的转座频率为22.7%.对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制--转座和不完全切离等现象.获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录.探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略.     

Saccharomyces cerevisiae Sae2- and Tel1-dependent single-strand DNA formation at DNA break promotes microhomology-mediated end joining

Microhomology-mediated end joining (MMEJ) joins DNA ends via short stretches [5-20 nucleotides (nt)] of direct repeat sequences, yielding deletions of intervening sequences. Non-homologous end joining (NHEJ) and single-strand annealing (SSA) are other error prone processes that anneal single-stranded DNA (ssDNA) via a few bases (<5 nt) or extensive direct repeat homologies (>20 nt). Although the genetic components involved in MMEJ are largely unknown, those in NHEJ and SSA are characterized in some detail. Here, we surveyed the role of NHEJ or SSA factors in joining of double-strand breaks (DSBs) with no complementary DNA ends that rely primarily on MMEJ repair. We found that MMEJ requires the nuclease activity of Mre11/Rad50/Xrs2, 3' flap removal by Rad1/Rad10, Nej1, and DNA synthesis by multiple polymerases including Pol4, Rad30, Rev3, and Pol32. The mismatch repair proteins, Rad52 group genes, and Rad27 are dispensable for MMEJ. Sae2 and Tel1 promote MMEJ but inhibit NHEJ, likely by regulating Mre11-dependent ssDNA accumulation at DNA break. Our data support the role of Sae2 and Tel1 in MMEJ and genome integrity.     

Regulation of activator/dissociation transposition by replication and DNA methylation

In maize the transposable elements Activator/Dissociation (Ac/Ds) transpose shortly after replication from one of the two resulting chromatids ("chromatid selectivity"). A model has been suggested that explains this phenomenon as a consequence of different affinity for Ac transposase binding to holo-, hemi-, and unmethylated transposon ends. Here we demonstrate that in petunia cells a holomethylated Ds is unable to excise from a nonreplicating vector and that replication restores excision. A Ds element hemi-methylated on one DNA strand transposes in the absence of replication, whereas hemi-methylation of the complementary strand causes a >6.3-fold inhibition of Ds excision. Consistently in the active hemi-methylated state, the Ds ends have a high binding affinity for the transposase, whereas binding to inactive ends is strongly reduced. These results provide strong evidence for the above-mentioned model. Moreover, in the absence of DNA methylation, replication enhances Ds transposition in petunia protoplasts >8-fold and promotes formation of a predominant excision footprint. Accordingly, replication also has a methylation-independent regulatory effect on transposition.     

Circularized Ac/Ds Transposons: Formation, Structure and Fate

The maize Ac/Ds transposable elements are thought to transpose via a cut-and-paste mechanism, but the intermediates formed during transposition are still unknown. In this work we present evidence that circular Ac molecules are formed in plants containing actively transposing elements. In these circles, transposon ends are joined head-to-head. The sequence at the ends' junction is variable, containing small deletions or insertions. Circles containing deleted Ac ends are probably unable to successfully reintegrate. To test the ability of circles with intact transposon ends to integrate into the genome, an artificial Ds circle was constructed by cloning the joined ends of Ac into a plasmid carrying a plant selectable marker. When such a circular Ds was introduced into tobacco protoplasts in the presence of Ac-transposase, no efficient transposase-mediated integration was observed. Although a circular transposition intermediate cannot be ruled out, the findings of circles with deleted transposon ends and the absence of transposase-mediated integration of the circular Ds suggest that some of the joined-ends-carrying elements are not transposition intermediates, but rather abortive excision products. The formation of Ac circles might account for the previously described phenomenon of Ac-loss. The origin of Ac circles and the implications for models of Ac transposition are discussed.     

Rad52 and Ku bind to different DNA structures produced early in double-strand break repair

DNA double-strand breaks are repaired by one of two main pathways, non-homologous end joining or homologous recombination. A competition for binding to DNA ends by Ku and Rad52, proteins required for non-homologous end joining and homologous recombination, respectively, has been proposed to determine the choice of repair pathway. In order to test this idea directly, we compared Ku and human Rad52 binding to different DNA substrates. How ever, we found no evidence that these proteins would compete for binding to the same broken DNA ends. Ku bound preferentially to DNA with free ends. Under the same conditions, Rad52 did not bind preferentially to DNA ends. Using a series of defined substrates we showed that it is single-stranded DNA and not DNA ends that were preferentially bound by Rad52. In addition, Rad52 aggregated DNA, bringing different single-stranded DNAs in close proximity. This activity was independent of the presence of DNA ends and of the ability of the single-stranded sequences to form extensive base pairs. Based on these DNA binding characteristics it is unlikely that Rad52 and Ku compete as ‘gatekeepers’ of different DNA double-strand break repair pathways. Rather, they interact with different DNA substrates produced early in DNA double-strand break repair.     

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.     

The fission yeast MRN complex tethers dysfunctional telomeres for NHEJ repair

Telomeres protect the natural ends of chromosomes from being repaired as deleterious DNA breaks. In fission yeast, absence of Taz1 (homologue of human TRF1 and TRF2) renders telomeres vulnerable to DNA repair. During the G1 phase, when non‐homologous end joining (NHEJ) is upregulated, taz1Δ cells undergo telomere fusions with consequent loss of viability. Here, we show that disruption of the fission yeast MRN (Rad23^MRE11‐Rad50‐Nbs1) complex prevents NHEJ at telomeres and, as a result, rescues taz1Δ lethality in G1. Neither Tel1^ATM activation nor 5′‐end resection was required for telomere fusion. Nuclease activity of Rad32^MRE11 was also dispensable for NHEJ. Mutants unable to coordinate metal ions required for nuclease activity were proficient in NHEJ repair. In contrast, Rad32^MRE11 mutations that affect binding and/or positioning of DNA ends leaving the nuclease function largely unaffected also impaired NHEJ at telomeres and restored the viability of taz1Δ in G1. Consistently, MRN structural integrity but not nuclease function is also required for NHEJ of independent DNA ends in a novel split‐molecule plasmid assay. Thus, MRN acts to tether unlinked DNA ends, allowing for efficient NHEJ.     

Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes.

S. cerevisiae RAD50, MRE11, and XRS2 genes are required for telomere maintenance, cell cycle checkpoint signaling, meiotic recombination, and the efficient repair of DNA double-strand breaks (DSB)s by homologous recombination and nonhomologous end-joining (NHEJ). Here, we demonstrate that the complex formed by Rad50, Mre11, and Xrs2 proteins promotes intermolecular DNA joining by DNA ligase IV (Dnl4) and its associated protein Lif1. Our results show that the Rad50/Mre11/Xrs2 complex juxtaposes linear DNA molecules via their ends to form oligomers and interacts directly with Dnl4/Lif1. We also demonstrate that Rad50/Mre11/Xrs2-mediated intermolecular DNA joining is further stimulated by Hdf1/Hdf2, the yeast homolog of the mammalian Ku70/Ku80 heterodimer. These studies reveal specific functional interplay among the Hdf1/Hdf2, Rad50/Mre11/Xrs2, and Dnl4/Lif1 complexes in NHEJ.     

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.     

Breadth by depth: expanding our understanding of the repair of transposon-induced DNA double strand breaks via deep-sequencing

The transposases of DNA transposable elements catalyze the excision of the element from the host genome, but are not involved in the repair of the resulting double-strand break. To elucidate the role of various host DNA repair and damage response proteins in the repair of the hairpin-ended double strand breaks (DSBs) generated during excision of the maize Ac element in Arabidopsis thaliana, we deep-sequenced hundreds of thousands of somatic excision products from a variety of repair- or response-defective mutants. We find that each of these repair/response defects negatively affects the preservation of the ends, resulting in an enhanced frequency of deletions, insertions, and inversions at the excision site. The spectra of the resulting repair products demonstrate, not unexpectedly, that the canonical nonhomologous end joining (NHEJ) proteins DNA ligase IV and KU70 play an important role in the repair of the lesion generated by Ac excision. Our data also indicate that auxiliary NHEJ repair proteins such as DNA ligase VI and DNA polymerase lambda are routinely involved in the repair of these lesions. Roles for the damage response kinases ATM and ATR in the repair of transposition-induced DSBs are also discussed.     

DNA双链断裂的非同源末端连接修复

细胞内普遍存在的DNA双链断裂(DSB)可通过同源重组(HR)或非同源末端连接(NHEJ)修复。由于HR仅在存在相同染色体作为模板的时候进行,因此,NHEJ通常为主要的修复方式。在NHEJ中,DSB末端首先由Ku识别,接着由核酸酶、聚合酶在Ku与DNA-PKcs协助下加工,并由连接酶IVXRCC4-XLF连接。NHEJ底物类型多样,末端的修复常包含反复加工的过程,导致修复产物通常无法复原损伤前的序列。虽然无法确保准确修复DNA,NHEJ仍对维持基因组的稳定性具有重要的意义。对NHEJ的研究有助于理解癌症的发生机制并将促进癌症的治疗。     

DNA homologous recombination repair in mammalian cells

DNA double-strand breaks (DSBs) are the most serious DNA damage. Due to a great variety of factors causing DSBs, the efficacy of their repair is crucial for the cell's functioning and prevents DNA fragmentation, chromosomal translocation and deletion. In mammalian cells DSBs can be repaired by non-homologous end joining (NHEJ), homologous recombination (HRR) and single strand annealing (SSA). HRR can be divided into the first and second phase. The first phase is initiated by sensor proteins belonging to the MRN complex, that activate the ATM protein which target HRR proteins to obtain the second response phase--repair. HRR is precise because it utilizes a non-damaged homologous DNA fragment as a template. The key players of HRR in mammalian cells are MRN, RPA, Rad51 and its paralogs, Rad52 and Rad54.     

Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination

DNA double strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The DNA cell cycle stage and resection of the DSB ends are two key mechanisms which are believed to push DSB repair to the HR pathway. Here, we show that the NHEJ factor Ku80 associates with DSBs in S phase, when HR is thought to be the preferred repair pathway, and its dynamics/kinetics at DSBs is similar to those observed for Ku80 in non-S phase in mammalian cells. A Ku homolog from Mycobacterium tuberculosis binds to and is retained at DSBs in S phase and was used as a tool to determine if blocking DNA ends affects end resection and HR in mammalian cells. A decrease in DNA end resection, as marked by IR-induced RPA, BrdU, and Rad51 focus formation, and HR are observed when Ku deficient rodent cells are complemented with Mt-Ku. Together, this data suggests that Ku70/80 binds to DSBs in all cell cycle stages and is likely actively displaced from DSB ends to free the DNA ends for DNA end resection and thus HR to occur.