Identification of a DNA nonhomologous end-joining complex in bacteria

In eukaryotic cells, double-strand breaks (DSBs) in DNA are generally repaired by the pathway of homologous recombination or by DNA nonhomologous end joining (NHEJ). Both pathways have been highly conserved throughout eukaryotic evolution, but no equivalent NHEJ system has been identified in prokaryotes. The NHEJ pathway requires a DNA end-binding component called Ku. We have identified bacterial Ku homologs and show that these proteins retain the biochemical characteristics of the eukaryotic Ku heterodimer. Furthermore, we show that bacterial Ku specifically recruits DNA ligase to DNA ends and stimulates DNA ligation. Loss of these proteins leads to hypersensitivity to ionizing radiation in Bacillus subtilis. These data provide evidence that many bacteria possess a DNA DSB repair apparatus that shares many features with the NHEJ system of eukarya and suggest that this DNA repair pathway arose before the prokaryotic and eukaryotic lineages diverged.     

Mycobacterial Ku and ligase proteins constitute a two-component NHEJ repair machine

In mammalian cells, repair of DNA double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ) is critical for genome stability. Although the end-bridging and ligation steps of NHEJ have been reconstituted in vitro, little is known about the end-processing reactions that occur before ligation. Recently, functionally homologous end-bridging and ligation activities have been identified in prokarya. Consistent with its homology to polymerases and nucleases, we demonstrate that DNA ligase D from Mycobacterium tuberculosis (Mt-Lig) possesses a unique variety of nucleotidyl transferase activities, including gap-filling polymerase, terminal transferase, and primase, and is also a 3' to 5' exonuclease. These enzyme activities allow the Mt-Ku and Mt-Lig proteins to join incompatible DSB ends in vitro, as well as to reconstitute NHEJ in vivo in yeast. These results demonstrate that prokaryotic Ku and ligase form a bona fide NHEJ system that encodes all the recognition, processing, and ligation activities required for DSB repair.     

A sense of the end

How a cell distinguishes a double-strand break from the end of a chromosome has long fascinated cell biologists. It was thought that the protection of chromosomal ends required either a telomere-specific complex or the looping back of the 3' TG-rich overhang to anneal with a homologous double-stranded repeat. These models must now accommodate the findings that complexes involved in nonhomologous end joining play important roles in normal telomere length maintenance, and that subtelomeric chromatin changes in response to the DNA damage checkpoint. A hypothetical chromatin assembly checkpoint may help to explain why telomeres and the double-strand break repair machinery share essential components.     

用CRISPR-Cas9在绵羊成纤维细胞ACTG1导入荧光标记基因

【目的】在绵羊成纤维细胞中,针对ACTG1基因羧基端,定点导入荧光蛋白标记基因,将外源基因定点导入绵羊基因组中,建立有效的方法。【方法】CRISPR-Cas9系统在绵羊成纤维细胞基因组特定区域引起DNA双链断裂,从而诱导细胞修复断裂的的基因组。通过NHEJ修复途径,在特定位点导入外源基因,改善定点导入效率。【结果】采用CRISPaint通用供体模板,结合CRISPR-Cas9系统,在绵羊成纤维细胞ACTG1基因导入荧光标记效率达1.4%,获得了外源基因定点导入的单克隆细胞株。【结论】CRISPR-Cas9系统结合NHEJ,能够有效的将大的外源DNA序列导入绵羊成纤维细胞的预定基因组位点。     

植物DNA双链断裂损伤修复机制研究进展

DNA双链断裂(DSBs)是细胞最严重的损伤形式之一。高等动植物中主要通过非同源末端连接（NHEJ）途径进行DNA双链断裂修复。该途径不依赖DNA同源性,由一些修复因子如：Ku蛋白异二聚体、DNA-PKcs 、XRCC4、ligaseⅣ等,将断裂末端直接连接进行修复。综述了植物DNA双链断裂损伤修复的主要途径及其相关基因研究的进展,探讨了植物DNA损伤修复研究中存在的问题与发展方向。     

SIRT6 promotes DNA repair under stress by activating PARP1

Sirtuin 6 (SIRT6) is a mammalian homolog of the yeast Sir2 deacetylase. Mice deficient for SIRT6 exhibit genome instability. Here, we show that in mammalian cells subjected to oxidative stress SIRT6 is recruited to the sites of DNA double-strand breaks (DSBs) and stimulates DSB repair, through both nonhomologous end joining and homologous recombination. Our results indicate that SIRT6 physically associates with poly[adenosine diphosphate (ADP)-ribose] polymerase 1 (PARP1) and mono-ADP-ribosylates PARP1 on lysine residue 521, thereby stimulating PARP1 poly-ADP-ribosylase activity and enhancing DSB repair under oxidative stress.     

StKU80, a component in the NHEJ repair pathway,is involved in mycelial morphogenesis,conidiation, appressorium development,and oxidative stress reactions in Exserohilum turcicum

Homologous recombination(HR) and nonhomologous end joining(NHEJ) are considered the two main double-strand break(DSB) repair approaches in eukaryotes. Inhibiting the activities of the key component in NHEJ commonly enhances the efficiency of targeted gene knockouts or affects growth and development in higher eukaryotes. However, little is known about the roles of the NHEJ pathway in foliar pathogens. Here we identified a gene designated St KU80, which encodes a putative DNA end-binding protein homologous to yeast Ku80, in the foliar pathogen Exserohilum turcicum. Conserved domain analysis showed that the typical domains VWA, Ku78 and Ku-PK-bind are usually present in Ku70/80 proteins in eukaryotes and are also present in St Ku80. Phylogenetic analysis indicated that St Ku80 is most closely related to Ku80(XP_001802136.1) from Parastagonospora nodorum, followed by Ku80(AGF90044.1) from Monascus ruber. Furthermore, the gene knockout mutants ΔSt KU80-1 and ΔSt KU80-2 were obtained. These mutants displayed longer septas, thinner cell walls, smaller amounts of substances on cell wall surfaces, and more mitochondria per cell than the wild-type(WT) strain but similar HT-toxin activity. The mutants did not produce conidia and mature appressoria. On the other hand, the mutants were highly sensitive to H_2O_2, but not to ultraviolet radiation. In summary, the St KU80 plays devious roles in regulating the development of E. turcicum.     

基于同源重组的 CRISPR 精准基因编辑技术 及其在农作物育种中的应用

随着 CRISPR 基因编辑技术的出现,几乎在任何动植物细胞基因组的特定目标位点,DNA 大片段 的“无缝”插入或替换,均可在 CRISPR 核酸酶产生双链切口后,在供体 DNA 存在的情况下,诱导同源定向修 复来实现。目前,这种基于同源重组的 CRISPR 精准基因编辑在农作物基因功能分析和新技术育种中正发挥着 越来越重要的作用。围绕在植物细胞中高效实现同源重组介导的 CRISPR 精准编辑这一目标,简述 CRISPR 精 准编辑依赖的两种主要的基于同源重组的细胞修复机制,即合成依赖的链退火修复机制和非同源末端连接辅助 的单链退火修复机制;在此基础上,详述产生 DNA 双链切口并诱导同源重组定向修复的 CRISPR 核酸酶和供体 DNA/RNA,主要包括 Cas9/12 及其融合蛋白、sgRNA/crRNA 及其修饰物、供体 DNA/RNA 及其修饰物;进而总 结在植物遗传转化中为保障 DNA 双链切口和供体 DNA/RNA 发生的时空一致性以提高同源重组效率,而通常采 用的 CRISPR 组分及供体 DNA/RNA 细胞递送方式;最后从功能基因组学研究和农作物新技术育种等方面,展望 基于同源重组的 CRISPR 精准基因编辑技术的应用前景。     

Cd胁迫诱导拟南芥幼苗DNA损伤分析

以拟南芥为供试植物,通过基于随机引物扩增多态性(RAPD)法的DNA损伤分析,酶联免疫吸附(ELISA)法的DNA甲基化分析以及Real-time PCR的DNA损伤修复与细胞周期相关基因的表达分析,研究了Cd(0、0.125、0.25、1.0、2.5 mg·L~(-1))胁迫5 d的拟南芥幼苗DNA损伤、DNA损伤修复系统以及细胞周期对胁迫的响应。结果显示,随Cd浓度的增加DNA损伤加剧,全基因组甲基化水平较对照组显著增加(P0.01或P0.05),细胞周期调控基因PCNA1、PCNA2,错配修复(MMR)基因MLH1、MSH_2、MSH6,非同源末端连接(NHEJ)标志基因KU70、MRE11、GR1,同源重组(HR)标志基因RAD51、BRCA1的表达均与Cd胁迫浓度呈明显的倒U型剂量效应关系,DNA修复系统对Cd胁迫的敏感性依次为MMRHRNHEJ。该结果表明:轻度Cd胁迫主要引起DNA错配损伤,并且该损伤易修复;随着Cd胁迫的增强,会引起DNA断裂与染色体损伤,损伤较难修复。另外,对Cd胁迫响应最敏感的MSH6、MLH1基因可作为表征Cd胁迫对于拟南芥遗传毒性效应的有效生物标记物。     

Inhibition of KU70 and KU80 by CRISPR interference,not NgAgo interference,increases the efficiency of homologous recombination in pig fetal fibroblasts

Non-homologous end-joining (NHEJ) is a predominant pathway for the repair of DNA double-strand breaks (DSB). It inhibits the efficiency of homologous recombination (HR) by competing for DSB targets. To improve the efficiency of HR, multiple CRISPR interference (CRISPRi) and Natronobacterium gregoryi Argonaute (NgAgo) interference (NgAgoi) systems have been designed for the knockdown of NHEJ key molecules, KU70, KU80, polynucleotide kinase/phosphatase (PNKP), DNA ligase IV (LIG4), and NHEJ1. Suppression of KU70 and KU80 by CRISPRi dramatically promoted (P<0.05) the efficiency of HR to 1.85- and 1.58-fold, respectively, whereas knockdown of PNKP, LIG4, and NHEJ1 repair factors did not significantly increase (P>0.05) HR efficiency. Interestingly, although the NgAgoi system significantly suppressed (P<0.05) KU70, KU80, PNKP, LIG4, and NHEJ1 expression, it did not improve (P>0.05) HR efficiency in primary fetal fibroblasts. Our result showed that both NgAgo and catalytically inactive Cas9 (dCas9) could interfere with the expression of target genes, but the downstream factors appear to be more active following CRISPR-mediated interference than that of NgAgo.     

Discovery of a predicted DNA knot substantiates a model for site-specific recombination

The mechanism of site-specific genetic recombination mediated by Tn3 resolvase has been investigated by a topological approach. Extrapolation of a detailed model of synapsis and strand exchange predicts the formation of an additional DNA product with a specific knotted structure. Two-dimensional gel electrophoresis of DNA reacted in vitro revealed a product, about 0.1 percent of the total, with the appropriate mobility. A technique for determining DNA topology by electron microscopy was improved such that less than a nanogram of DNA was required. The structure of the knot was as predicted, providing strong evidence for the model and showing the power of the topological method.     

ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex

The ataxia-telangiectasia mutated (ATM) kinase signals the presence of DNA double-strand breaks in mammalian cells by phosphorylating proteins that initiate cell-cycle arrest, apoptosis, and DNA repair. We show that the Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules. Inactive ATM dimers were activated in vitro with DNA in the presence of MRN, leading to phosphorylation of the downstream cellular targets p53 and Chk2. ATM autophosphorylation was not required for monomerization of ATM by MRN. The unwinding of DNA ends by MRN was essential for ATM stimulation, which is consistent with the central role of single-stranded DNA as an evolutionarily conserved signal for DNA damage.     

A conserved checkpoint monitors meiotic chromosome synapsis in Caenorhabditis elegans

We report the discovery of a checkpoint that monitors synapsis between homologous chromosomes to ensure accurate meiotic segregation. Oocytes containing unsynapsed chromosomes selectively undergo apoptosis even if a germline DNA damage checkpoint is inactivated. This culling mechanism is specifically activated by unsynapsed pairing centers, cis-acting chromosome sites that are also required to promote synapsis in Caenorhabditis elegans. Apoptosis due to synaptic failure also requires the C. elegans homolog of PCH2, a budding yeast pachytene checkpoint gene, which suggests that this surveillance mechanism is widely conserved.     

Removal of shelterin reveals the telomere end-protection problem

The telomere end-protection problem is defined by the aggregate of DNA damage signaling and repair pathways that require repression at telomeres. To define the end-protection problem, we removed the whole shelterin complex from mouse telomeres through conditional deletion of TRF1 and TRF2 in nonhomologous end-joining (NHEJ) deficient cells. The data reveal two DNA damage response pathways not previously observed upon deletion of individual shelterin proteins. The shelterin-free telomeres are processed by microhomology-mediated alternative-NHEJ when Ku70/80 is absent and are attacked by nucleolytic degradation in the absence of 53BP1. The data establish that the end-protection problem is specified by six pathways [ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related) signaling, classical-NHEJ, alt-NHEJ, homologous recombination, and resection] and show how shelterin acts with general DNA damage response factors to solve this problem.     

Three-dimensional structure of the Tn5 synaptic complex transposition intermediate

Genomic evolution has been profoundly influenced by DNA transposition, a process whereby defined DNA segments move freely about the genome. Transposition is mediated by transposases, and similar events are catalyzed by retroviral integrases such as human immunodeficiency virus-1 (HIV-1) integrase. Understanding how these proteins interact with DNA is central to understanding the molecular basis of transposition. We report the three-dimensional structure of prokaryotic Tn5 transposase complexed with Tn5 transposon end DNA determined to 2.3 angstrom resolution. The molecular assembly is dimeric, where each double-stranded DNA molecule is bound by both protein subunits, orienting the transposon ends into the active sites. This structure provides a molecular framework for understanding many aspects of transposition, including the binding of transposon end DNA by one subunit and cleavage by a second, cleavage of two strands of DNA by a single active site via a hairpin intermediate, and strand transfer into target DNA.     

Tumor DNA structure in plant cells transformed by A. tumefaciens

Crown gall tumors are induced in plants by infection with the soil bacterium Agrobacterium tumefaciens. Because the tumor induction involves transfer of a portion of the tumor-inducing (Ti) plasmid DNA from the bacterium to the plant cells, this system is of interest for the study of genetic exchange as well as tumor induction. The boundaries of the transferred DNA (T-DNA) have been cloned from transformed plant cells of tobacco. Detailed mapping with restriction enzymes and nucleotide sequence analysis of two independent clones were used to study the molecular structure of the ends of the T-DNA. One clone contains the two ends of the T-DNA joined together; the other contains one end of the T-DNA joined to repetitive plant DNA sequences. These studies provide direct evidence that the T-DNA can be integrated into the plant genome. In addition, the data suggest that in the plant, T-DNA can be tandemly repeated. Sequence analysis of the junction of crown gall clone 1 reveals several direct repeats as well as an inverted repeat; these structures may be involved in the transfer of the DNA from Agrobacterium to plant cells.     

The t(14;18) chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining

In this study, the joining sequences between chromosomes 14 and 18 on the 14q+ chromosomes of a patient with pre-B-cell leukemia and four patients with follicular lymphoma carrying a t(14;18) chromosome translocation were analyzed. In each case, the involved segment of chromosome 18 has recombined with the immunoglobulin heavy-chain joining segment (JH) on chromosome 14. The sites of the recombination on chromosome 14 are located close to the 5' end of the involved JH segment, where the diversity (D) regions are rearranged with the JH segments in the production of active heavy-chain genes. As extraneous nucleotides (N regions) were observed at joining sites and specific signal-like sequences were detected on chromosome 18 in close proximity to the breakpoints, it is concluded that the t(14;18) chromosome translocation is the result of a mistake during the process of VDJ joining at the pre-B-cell stage of differentiation. The putative recombinase joins separated DNA segments on two different chromosomes instead of joining separated segments on the same chromosome, causing a t(14;18) chromosome translocation in the involved B cells.     

5'-Deoxyribose phosphate lyase activity of human DNA polymerase iota in vitro

DNA polymerase iota (pol iota) is one of several recently discovered DNA polymerases in mammalian cells whose function is unknown. We report here that human pol iota has an intrinsic 5'-deoxyribose phosphate (dRP) lyase activity. In reactions reconstituted with uracil-DNA glycosylase (UDG), apurinic/apyrimidinic (AP) endonuclease and DNA ligase I, pol iota can use its dRP lyase and polymerase activities to repair G*U and A*U pairs in DNA. These data and three distinct catalytic properties of pol iota implicate it in specialized forms of base excision repair (BER).