DNA end joining activity is reduced in Alzheimer's disease

Evidence indicates that oxidative stress-induced damage to DNA, protein, and other cellular components contributes to the progression of Alzheimer's disease (AD). Several studies indicate that postmitotic neurons have a reduced capacity for some types of DNA repair, which is further compromised by aging. Thus in AD, the cellular response to increased oxidative DNA damage may be inadequate to protect the genome. Mammalian cells use several mechanisms to repair DNA damage generated during normal oxidative metabolism or by genotoxic insults. The predominant mechanism to repair double strand breaks is non-homologous end joining (NHEJ) which utilizes the DNA-dependent protein kinase (DNA-PK) complex. A cell-free DNA end joining assay was employed to determine if NHEJ was reduced in nuclear cortical extracts from brains of AD versus normal subjects. This report demonstrates that end joining activity and protein levels of DNA-PK catalytic subunit are significantly lower in AD brains compared to normal controls. The amount of end joining activity correlates with the expression of DNA-PK and is dependent on DNA-PK catalytic activity. This indicates that repair of DNA double-strand breaks by the DNA-PK-dependent NHEJ pathway may be deficient in AD.     

DNA end sequestration by DNA-dependent protein kinase and end joining of sterically constrained substrates in whole-cell extracts

Extracts of Xenopus eggs and of cultured human and hamster cells have the capacity to join nonhomologous DNA ends, and all do so with similar specificity. To examine the formation of repair complexes on DNA under conditions of end joining, end-labeled fragments were incubated with the various extracts and then subjected to DNase-I footprinting. Human and Xenopus extracts produced footprints virtually identical to that of purified DNA-dependent protein kinase holoenzyme (Ku plus DNA-PKcs), with protection of the terminal 28 bp. Extracts of hamster cells were more variable, but usually produced a 16-bp footprint, similar to that of Ku alone. In all cases a 28-bp holoenzyme-like footprint was associated with wortmannin-sensitive end joining, minimal 3'-5' exonucleolytic resection, and a predominance of accurate end-joining products. To determine whether the short segments of DNA occupied by Ku and DNA-PK were sufficient to support end joining, Y-shaped substrates were constructed in which only one arm was available for end joining. A Y substrate with a 31-bp arm bearing a partially cohesive 3' overhang was accurately joined by a Xenopus egg extract, whereas a substrate with a 21-bp arm was not. Surprisingly, a human cell extract did not join the Y substrates at all. The results suggest that differences in wortmannin sensitivity and in the distribution of in vitro end-joining products may be attributable to the variations in the levels of DNA-PKcs in the extracts. In addition, end joining in human extracts appears to involve interactions with significantly longer segments of DNA than the approximately 28 bp occupied by DNA-PK.     

Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans

DNA lesions that block replication fork progression are drivers of cancer-associated genome alterations, but the error-prone DNA repair mechanisms acting on collapsed replication are incompletely understood, and their contribution to genome evolution largely unexplored. Here, through whole-genome sequencing of animal populations that were clonally propagated for more than 50 generations, we identify a distinct class of deletions that spontaneously accumulate in C. elegans strains lacking translesion synthesis (TLS) polymerases. Emerging DNA double-strand breaks are repaired via an error-prone mechanism in which the outermost nucleotide of one end serves to prime DNA synthesis on the other end. This pathway critically depends on the A-family polymerase theta, which protects the genome against gross chromosomal rearrangements. By comparing the genomes of isolates of C. elegans from different geographical regions, we found that in fact most spontaneously evolving structural variations match the signature of polymerase theta-mediated end joining (TMEJ), illustrating that this pathway is an important source of genetic diversification.Identifying the mechanisms that fuel genome change is crucial for understanding evolution and carcinogenesis. Spontaneous mutagenesis is caused predominantly by misinsertions or slippage events of replicative polymerases that are missed by their proofreading domains and not corrected by mismatch repair (Lynch 2008). Less frequently, but with a potentially much more detrimental effect, mutations can arise when DNA damage obstructs progression of DNA replication; and stalled replication forks eventually collapse, resulting in highly mutagenic double-stranded breaks (DSBs). Although error-free homologous repair, in which the sister chromatid is used as a template, restores the original sequence, infrequent but highly mutagenic error-prone end joining processes can give rise to spontaneous deletions and tumor-promoting translocations (Mitelman et al. 2007).To circumvent fork collapse at DNA damage, cells use various alternative polymerases that are capable of incorporating nucleotides across DNA lesions and are hence called translesion synthesis (TLS) polymerases. TLS acts on a wide variety of DNA lesions that can result from endogenous as well as exogenous genotoxic sources: DNA lesions that result from UV-light exposure, for instance, are efficiently bypassed by the well-conserved TLS polymerase eta (pol eta), inactivation of which in humans leads to the variant form of the skin cancer predisposition syndrome, Xeroderma Pigmentosum (Masutani et al. 1999b; Johnson et al. 2007). Abundant in vitro studies demonstrate the involvement of TLS polymerases pol eta and pol kappa in the bypass of lesions that are produced by endogenous reactive compounds, showing that these polymerases are also essential for protection of the genome under unchallenged conditions (Haracska et al. 2000; Fischhaber et al. 2002; Kusumoto et al. 2002).Although error-prone while replicating, and thus potentially causing misinsertions, TLS polymerases are thought to protect cells against the more mutagenic effects of replication fork collapse (Knobel and Marti 2011). Here, we investigate the contribution of TLS polymerases on the maintenance of genome stability and the mechanisms acting on stalled DNA replication by characterizing C. elegans strains that are defective for the Y-family polymerases pol eta and pol kappa. Unexpectedly, we found that DSBs resulting from replication-blocking endogenous lesions are not repaired via canonical DSB repair pathways, but through an error-prone repair mechanism that critically depends on the A-family DNA polymerase theta (pol theta).     

DNA非同源末端连接与肿瘤的发生及治疗

DNA双链断裂是真核生物最严重也是最致命的DNA损伤类型,如果无法及时修复就可能启动细胞的凋亡,若是错误修复将导致基因突变或基因组不稳定性,最终会导致肿瘤的产生.非同源末端连接途径作为哺乳动物修复双链断裂的主要机制,它的功能不仅对维持基因组的完整性和细胞的稳定性有重要作用,而且和肿瘤治疗疗效有着密切相关性,修复活性的抑制可以很大程度地提高治疗的敏感性.该文综述了非同源末端连接途径在肿瘤的发生及治疗上的重要作用和机制.     

Normal human oral keratinocytes demonstrate abnormal DNA end joining activity during replicative senescence

Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of cellular genetic integrity. DSBs are repaired by cellular end joining activity, which could proceed with varying degrees of accuracy. Abnormal end joining may lead to an accumulation of mutations and contribute to genetic instability and cellular aging. In the present study, we compared the efficiency and accuracy of end joining activities in exponentially replicating and senescing normal human oral keratinocytes (NHOK). We developed an in vitro end joining assay utilizing a plasmid linearized with a unique EcoR I or EcoR V restriction site. The efficiency of end joining was determined by PCR with primers that could amplify the fragment containing the end joining site. The accuracy of end joining was assessed by determining whether the original EcoR I site was restored after end joining. Both replicating and senescing cultures of NHOK yielded a similar level of end joining efficiency, which was noted by the similar intensity of PCR amplification. However, the frequency of end joining errors was significantly elevated in NHOK during replicative senescence. Senescing NHOK could thus accumulate abnormal end joining products, which might contribute to cellular aging and cancer.     

The biochemistry and biological significance of nonhomologous DNA end joining: an essential repair process in multicellular eukaryotes

Recent progress over the past year has provided new insights into the proteins involved in nonhomologous end joining. The assembly of Ku and DNA-dependent protein kinase at DNA ends is now understood in greater detail. Murine genetic knockouts for DNA ligase IV and XRCC4 are embryonic lethal, indicating that nonhomologous end joining is essential for viability. Interestingly, neurones, in addition to lymphocytes, are particularly vulnerable to an absence of NHEJ.     

Alternative end joining during switch recombination in patients with ataxia-telangiectasia

Ataxia-Telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are recessive genetic diseases with similar cellular phenotypes that are caused by mutations in the recently described ATM (encoding ATM) and NBS1 (encoding p95) genes, respectively. Both disorders are accompanied by immunodeficiency in a majority of patients, but the mechanism involved has as yet not been established. We demonstrate that in cells from A-T patients, the switch (S) recombination junctions are aberrant and characterized by a strong dependence on short sequence homologies and devoid of normally occurring mutations around the breakpoint. A low number of S fragments were generated in cells from NBS patients and showed only limited dependence on sequence identity and mutation frequencies were similar to those observed in normal controls. We propose that ATM and p95 are both involved in the final step(s) in class switch recombination with related, but disparate, functional roles. Thus, the general pathway involved in DNA repair also has a major influence on the immunoglobulin isotype switching process.     

非同源末端连接机制与基因扩增关联的研究进展

基因扩增是肿瘤中基因组异常形式之一,与肿瘤发生发展及耐药性有密切关系.非同源末端连接(non-homologous end joining,NHEJ)是哺乳动物DNA双链断裂(doublestrand breaks,DSBs)修复的主要机制之一,在维持基因组的稳定性中发挥着重要作用.近年来,非同源末端连接与基因扩增关系的研究受到越来越多的关注.本文分别介绍了基因扩增,两种非同源末端连接修复方式机制及其与基因扩增的研究进展.     

Restoration to the parental genotype of mismatches formed in recombinant DNA heteroduplex

Summary Crosses were set up, using the b2 locus of Ascobolus immersus, to detect and measure the restoration of the parental genotype of a chromatid from hetero-duplex. From the results it can be concluded that: (1) restoration occurs, and its frequency is comparable to the frequency of conversion; (2) the relative frequency of conversion and restoration on the two homologous chromatids is that expected if the disparity in conversion to mutant and to wild-type of the marker is caused by the decision whether to excise the mutant or wild-type chain. This decision is the same on both chromatids. However, when all data are pooled, a slight, but significant, excess of conversion over restoration is found. The cause of this in discussed; (3) the absence of an excess of restoration over conversion is taken to imply that, in this system, the length of heteroduplex formed in an event is continuous; (4) these findings support the interpretation of tetrads where a crossover is separated from conversion, that they arise by independent correction giving both conversion and restoration in a heteroduplex tract continuous with the crossover.     

Resolution of inflammation: the beginning programs the end

Acute inflammation normally resolves by mechanisms that have remained somewhat elusive. Emerging evidence now suggests that an active, coordinated program of resolution initiates in the first few hours after an inflammatory response begins. After entering tissues, granulocytes promote the switch of arachidonic acid-derived prostaglandins and leukotrienes to lipoxins, which initiate the termination sequence. Neutrophil recruitment thus ceases and programmed death by apoptosis is engaged. These events coincide with the biosynthesis, from omega-3 polyunsaturated fatty acids, of resolvins and protectins, which critically shorten the period of neutrophil infiltration by initiating apoptosis. Consequently, apoptotic neutrophils undergo phagocytosis by macrophages, leading to neutrophil clearance and release of anti-inflammatory and reparative cytokines such as transforming growth factor-beta1. The anti-inflammatory program ends with the departure of macrophages through the lymphatics. Understanding these and further details of the mechanism required for inflammation resolution may underpin the development of drugs that can resolve inflammatory processes in directed and controlled ways.     

Ku acts in a unique way at the mammalian telomere to prevent end joining

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusion events and to distinguish the chromosome ends from double-strand breaks. We report that TRF1 and Ku form a complex at the telomere. The Ku and TRF1 complex is a specific high-affinity interaction, as demonstrated by several in vitro methods, and exists in human cells as determined by coimmunoprecipitation experiments. Ku does not bind telomeric DNA directly but localizes to telomeric repeats via its interaction with TRF1. Primary mouse embryonic fibroblasts that are deficient for Ku80 accumulated a large percentage of telomere fusions, establishing that Ku plays a critical role in telomere capping in mammalian cells. We propose that Ku localizes to internal regions of the telomere via a high-affinity interaction with TRF1. Therefore, Ku acts in a unique way at the telomere to prevent end joining.     

Robust immunoglobulin class switch recombination and end joining in Parp9‐deficient mice

To mount highly specific and adapted immune responses, B lymphocytes assemble and diversify their antibody repertoire through mechanisms involving the formation of programmed DNA damage. Immunoglobulin class switch recombination (CSR) is triggered by DNA lesions induced by activation‐induced cytidine deaminase, which are processed to double‐stranded DNA break (DSB) intermediates. These DSBs activate the cellular DNA damage response and enroll numerous DNA repair factors, involving poly(ADP‐ribose) polymerases Parp1, Parp2, and Parp3 to promote appropriate DNA repair and efficient long‐range recombination. The macroParp Parp9, which is overexpressed in certain lymphomas, has been recently implicated in DSB repair, acting together with Parp1. Here, we examine the contribution of Parp9 to the resolution of physiological DSBs incurred during V(D)J recombination and CSR by generating Parp9^?/? mice. We find that Parp9‐ deficient mice are viable, fertile, and do not show any overt phenotype. Moreover, we find that Parp9 is dispensable for B‐cell development. Finally, we show that CSR and DNA end‐joining are robust in the absence of Parp9, indicating that Parp9 is not essential in vivo to achieve physiological DSB repair, or that strong compensatory mechanisms exist.     

Obligate Ligation-Gated Recombination (ObLiGaRe): Custom-designed nuclease-mediated targeted integration through nonhomologous end joining

Custom-designed nucleases (CDNs) greatly facilitate genetic engineering by generating a targeted DNA double-strand break (DSB) in the genome. Once a DSB is created, specific modifications can be introduced around the breakage site during its repair by two major DNA damage repair (DDR) mechanisms: the dominant but error-prone nonhomologous end joining (NHEJ) pathway, and the less-frequent but precise homologous recombination (HR) pathway. Here we describe ObLiGaRe, a new method for site-specific gene insertions that uses the efficient NHEJ pathway and acts independently of HR. This method is applicable with both zinc finger nucleases (ZFNs) and Tale nucleases (TALENs), and has enabled us to insert a 15-kb inducible gene expression cassette at a defined locus in human cell lines. In addition, our experiments have revealed the previously underestimated error-free nature of NHEJ and provided new tools to further characterize this pathway under physiological and pathological conditions.The development of custom-designed nucleases (CDNs), including zinc finger nucleases (ZFNs) and Tale nucleases (TALENs), has made it possible to perform precise genetic engineering in many cell types and species (Kim et al. 1996; Bibikova et al. 2003; Porteus and Baltimore 2003; Moehle et al. 2007; Hockemeyer et al. 2009; Christian et al. 2010; Meyer et al. 2010; Urnov et al. 2010). CDNs are hybrid endonucleases consisting of a FokI nuclease domain and a DNA binding domain assembled from optimized DNA binding modules that are specific for either single-nucleotide (for TALENs) or trinucleotide motifs (for ZFNs). Once introduced into cells, CDNs generate a double-strand break (DSB) in the genome at or near the desired modification site and induce DNA damage repair (DDR) to mend the break (Rouet et al. 1994). Repair is largely accomplished by error-prone nonhomologous end joining (NHEJ), in which the two ends are processed and ligated together in a way that is frequently accompanied by nucleotide insertions and deletions. Though highly efficient, NHEJ produces knockout alleles that are often heterogeneous, and individual cell clones must be isolated for characterization. Currently, specific gene modification relies on homologous recombination (HR), in which exogenous DNA fragments flanked by homologous sequences around the DSB site are copied faithfully from a template with defined boundaries (Rouet et al. 1994).We have successfully applied ZFNs to generate knockout and knock-in alleles directly in mouse zygotes (Meyer et al. 2010; Cui et al. 2011). While optimizing gene targeting conditions, we observed that a donor plasmid can be “ligated” into the genome if it contained the same ZFN recognition site as the targeted genomic locus. It has been reported that short, double-stranded DNAs with 5′ overhangs could be ligated to complementary ends generated after ZFN digestion (Orlando et al. 2010). This observation has not been further explored, probably because it requires the knowledge of the overhangs generated by ZFNs, and only insertions of small oligonucleotide have been described (Orlando et al. 2010). Furthermore, it has also been shown that donor molecules, including single-strand oligodeoxynucleotides (ssODNs) (Radecke et al. 2010; Chen et al. 2011) and larger external linear sequences, can be captured at DSB sites generated by ZFNs (Mittelman et al. 2009; Fung and Weinstock 2011; Gabriel et al. 2011; Li et al. 2011). This feature has been harnessed to track “off-target” effects of the homing endonuclease I-SceI and ZFNs (Petek et al. 2010). Based on these reports and our own observation in mouse embryos, we surmised that it should be possible to directly ligate an external DNA fragment linearized in situ by the same ZFNs that target the genome.We took advantage of the obligated heterodimeric property of the CDNs (Miller et al. 2007; Szczepek et al. 2007; Doyon et al. 2011; Ramalingam et al. 2011) and designed a strategy to achieve efficient and precise gene targeting without homology in the donor plasmid. We named this method ObLiGaRe (Obligate Ligation-Gated Recombination) to reflect the etymologic meaning of the Latin verb obligare (“to bind,” “to join to”). ObLiGaRe should be broadly applicable across different cell types and provides an additional approach for genetic engineering.     

Fancd2 functions in a double strand break repair pathway that is distinct from non-homologous end joining

Fanconi anemia (FA) is a multigenic recessive disease resulting in bone marrow failure and increased cancer susceptibility. Cells from FA patients and mouse models are sensitive to DNA interstrand crosslinks (ICLs) and FA mice are moderately sensitive to ionizing radiation (IR). Both kinds of damage induce DNA double strand breaks (DSBs). To date, nine genes in 11 complementation groups have been identified; however, the precise function of the FA pathway remains unclear. Many of the proteins form a nuclear complex necessary for the mono-ubiquitination of the downstream protein, Fancd2. To further investigate the role of the FA pathway in repair of DSBs, we generated Fancd2(-/-)/Prkdc(sc/sc) double mutant mice. Prkdc(sc/sc) mutant mice have a defect in non-homologous end joining (NHEJ) and are sensitive to IR-induced DNA damage. Double mutant animals and primary cells were more sensitive to IR than either single mutant, suggesting that Fancd2 operates in DSB repair pathway distinct from NHEJ. Fancd2(-/-)/Prkdc(sc/sc) double mutant cells were also more sensitive to DSBs generated by a restriction endonuclease. The role of Fancd2 in DSB repair may account for the moderate sensitivity of FA cells to irradiation and FA cells sensitivity to ICLs that are repaired via a DSB intermediate.     

LCR-initiated rearrangements at the IDS locus, completed with Alu-mediated recombination or non-homologous end joining

Mucopolysaccharidosis type II (MPS II) is caused by mutations in the IDS gene, which encodes the lysosomal enzyme iduronate-2-sulfatase. In ～20% of MPS II patients the disorder is caused by gross IDS structural rearrangements. We identified two male cases harboring complex rearrangements involving the IDS gene and the nearby pseudogene, IDSP1, which has been annotated as a low-copy repeat (LCR). In both cases the rearrangement included a partial deletion of IDS and an inverted insertion of the neighboring region. In silico analyses revealed the presence of repetitive elements as well as LCRs at the junctions of rearrangements. Our models illustrate two alternative consequences of rearrangements initiated by non-allelic homologous recombination of LCRs: resolution by a second recombination event (that is, Alu-mediated recombination), or resolution by non-homologous end joining repair. These complex rearrangements have the potential to be recurrent and may be present among those MSP II cases with previously uncharacterized aberrations involving IDS.     

Integrative recombination between adenovirus type 12 DNA and mammalian DNA in a cell-free system: joining by short sequence homologies

A cell-free system was developed to investigate the mechanism of how junctions are formed between viral and cellular DNAs during adenoviral DNA integration into the hamster cell genome. Recombination between the segment of adenovirus type 12 (Ad12) DNA, that comprises sequence coordinates 20 885–24 053, subsequently termed PstI-D fragment and the hamster preinsertion DNA sequence p7 was studied in a cell-free system. The p7 DNA segment had served as viral DNA integration site in the Ad12-induced tumor CLAC1. Nuclear extracts initially from uninfected BHK21 hamster cells were fractionated by a series of chromatographic steps. DNAs of the in vitro generated recombinants were analyzed in detail. In the course of the recombination reaction, the two linear molecules were joined. The reaction took place between two short homologous sequences one of which was always at or very close to a DNA terminus, the second one could be several kilobase pairs remote from a DNA terminus. Apparently, the nucleotide sequence at the terminus of one recombining molecule determined the point of junction by searching for short homologies in the partner molecule. The recombination reaction was not conservative, the sequences in-between the short sequence homologies and one of the short sequence homologies were deleted in the in vitro recombinants. Two main criteria influenced the choice of interacting short sequence homologies: perfect homologies of 8–9 bp were most frequently found, they were preferred over more extended, but less perfect homologies. Comparing different short sequence homologies with similar stabilities, those combinations seemed to be chosen in the reaction which led to a minimal loss of nucleotides in the recombinants. The in vitro activity was found in nuclear extracts from both hamster and human cells. The activity was, hence, available for Ad12 DNA in productively infected human and abortively infected hamster cells. The specific recombination activity was increased in nuclear extracts of hamster cells abortively infected with Ad12. The junction sites in the recombinants, which were generated by the cell-free system, were very similar to junctions between adenoviral and cellular DNAs cloned from Ad12-induced tumor cells and Ad12-transformed cell lines.     

A mathematically designed STS primer without any mismatches for direct sequencing of cosmid DNA clones

Summary We report here a new method for the direct sequencing of large DNA inserts of cosmid clones from human chromosomes using STS primers of 14 nucleotides without any mismatches, which are designed from results of a mathematical analysis. It is clear that STS primer of 14 nucleotides is optimum for direct sequencing of cosmid recombinant DNA clones. We also provide examples of direct sequencing of cosmid clones of human chromosome 21 using these STS primers.