Innate DNA sensing moves to the nucleus

It has been assumed that cells distinguish viral from cellular DNA due to the former's presence in the cytosol. However, in this issue, Kerur et al. (2011) propose that the DNA genome of Kaposi's sarcoma-associated herpesvirus (KSHV) is recognized inside the nucleus by the DNA sensor IFI16, leading inflammasome activation.     

A lncRNA to repair DNA

Long non‐coding RNAs (lncRNAs) have emerged as regulators of various biological processes, but to which extent lncRNAs play a role in genome integrity maintenance is not well understood. In this issue of EMBO Reports, Sharma et al 1 identify the DNA damage‐induced lncRNA DDSR1 as an integral player of the DNA damage response (DDR). DDSR1 has both an early role by modulating repair pathway choices, and a later function when it regulates gene expression. Sharma et al 1 thus uncover a dual role for a hitherto uncharacterized lncRNA during the cellular response to DNA damage.     

DNA repair pathway genes and lung cancer susceptibility: A meta-analysis

Objective

DNA repair pathway genes have been implicated to play an important role in the development of lung cancer. However, contradictory results are often reported by various studies, making it difficult to interpret them. So in this meta-analysis, we have assessed the association between lung cancer risk and two DNA repair pathway genes. XRCC1 and ERCC2, by analyzing 67 published case–control studies.

Research design and methods

We searched PubMed, Embase and Web of Science using terms “XRCC1” or “XPD” or “ERCC2” and “lung cancer” on August 1, 2012. Three criteria were applied to select included studies for resulting studies. Information was carefully extracted by two investigators independently. We used pooled odds ratio (OR) to assess the effect of a polymorphism, and a dominant model was applied where genotypes that contain the non-reference allele were combined together. All the calculations were performed using STATA version 11.0.

Main outcome measures and results

Three common nonsynonymous polymorphisms in XRCC1, codon 194, codon 280 and codon 399, and two common nonsynonymous polymorphisms in ERCC2, codon 312 and codon 751, were analyzed. The result showed in total population, Lys751Gln in ERCC2 is associated with an increase of lung cancer risk, with a summary OR as 1.15. No association was found for any other polymorphisms. When studies were stratified by ethnicity, the risk effect of Lys751Gln in ERCC2 was found only in Caucasians, not in Asians.

Conclusions

In conclusion, Lys751Gln in ERCC2 is associated with lung cancer, and the risk effect probably exists in Caucasians. By contrast, polymorphisms in XRCC1 are less likely to be susceptible to lung cancer risks.     

Regulation of DNA double-strand break repair pathway choice

DNA double-strand breaks （DSBs） are critical lesions that can result in cell death or a wide variety of genetic alterations including largeor small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining （NHEJ） and homologous recombination （HR）, and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1（XRS2） complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit （DNA-PKcs）, and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.     

The Fanconi anemia pathway and the DNA interstrand cross-links repair

Fanconi anemia (FA) is a genetic cancer-predisposition syndrome characterized by bone marrow failure and cellular and chromosomal hypersensitivity to DNA cross-linking agents. Seven FA genes have been isolated and their products associate to form a pathway that interacts functionally or physically with several DNA-damage response proteins involved in cell cycle checkpoints and/or DNA repair. These proteins include BLM, ATM, BRCA1, XPF and the MRE11/RAD50/NBS1 complex. In spite of several recent striking progresses in the biochemistry and the molecular biology of the disorder, the precise function(s) of the FA proteins remain(s) poorly determined. However, several recent data indicate that the FA pathway could be involved in the coordination of both cell cycle checkpoints and DNA repair.     

Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway

The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.     

Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery

The p53 tumor suppressor that plays a central role in the cellular response to genotoxic stress was suggested to be associated with the DNA repair machinery which mostly involves nucleotide excision repair (NER). In the present study we show for the first time that p53 is also directly involved in base excision repair (BER). These experiments were performed with p53 temperature-sensitive (ts) mutants that were previously studied in in vivo experimental models. We report here that p53 ts mutants can also acquire wild-type activity under in vitro conditions. Using ts mutants of murine and human origin, it was observed that cell extracts overexpressing p53 exhibited an augmented BER activity measured in an in vitro assay. Depletion of p53 from the nuclear extracts abolished this enhanced activity. Together, this suggests that p53 is involved in more than one DNA repair pathway.     

Regulation of double-stranded DNA gap repair by the RAD6 pathway

The RAD6 pathway allows replication across DNA lesions by either an error-prone or error-free mode. Error-prone replication involves translesion polymerases and requires monoubiquitylation at lysine (K) 164 of PCNA by the Rad6 and Rad18 enzymes. By contrast, the error-free bypass is triggered by modification of PCNA by K63-linked polyubiquitin chains, a reaction that requires in addition to Rad6 and Rad18 the enzymes Rad5 and Ubc13-Mms2. Here, we show that the RAD6 pathway is also critical for controlling repair pathways that act on DNA double-strand breaks. By using gapped plasmids as substrates, we found that repair in wild-type cells proceeds almost exclusively by homology-dependent repair (HDR) using chromosomal DNA as a template, whereas non-homologous end-joining (NHEJ) is suppressed. In contrast, in cells deficient in PCNA polyubiquitylation, plasmid repair occurs largely by NHEJ. Mutant cells that are completely deficient in PCNA ubiquitylation, repair plasmids by HDR similar to wild-type cells. These findings are consistent with a model in which unmodified PCNA supports HDR, whereas PCNA monoubiquitylation diverts repair to NHEJ, which is suppressed by PCNA polyubiquitylation. More generally, our data suggest that the balance between HDR and NHEJ pathways is crucially controlled by genes of the RAD6 pathway through modifications of PCNA.     

Ploidy dictates repair pathway choice under DNA replication stress

This study reports an unusual ploidy-specific response to replication stress presented by a defective minichromosome maintenance (MCM) helicase allele in yeast. The corresponding mouse allele, Mcm4(Chaos3), predisposes mice to mammary gland tumors. While mcm4(Chaos3) causes replication stress in both haploid and diploid yeast, only diploid mutants exhibit G2/M delay, severe genetic instability (GIN), and reduced viability. These different outcomes are associated with distinct repair pathways adopted in haploid and diploid mutants. Haploid mutants use the Rad6-dependent pathways that resume stalled forks, whereas the diploid mutants use the Rad52- and MRX-dependent pathways that repair double strand breaks. The repair pathway choice is irreversible and not regulated by the availability of repair enzymes. This ploidy effect is independent of mating type heterozygosity and not further enhanced by increasing ploidy. In summary, a defective MCM helicase causes GIN only in particular cell types. In response to replication stress, early events associated with ploidy dictate the repair pathway choice. This study uncovers a fundamental difference between haplophase and diplophase in the maintenance of genome integrity.     

DNA repair pathway choice—a PTIP of the hat to 53BP1

In the June issue of Cell, Nussenzweig and colleagues identify PTIP/PAXIP as a 53BP1 effector protein in the regulatory network that controls DSB repair pathway choice.Cell (2013) 153 6, 1266–1280 doi: 10.1016/j.cell.2013.05.023DNA double-stranded breaks (DSBs) are highly cytotoxic lesions that can induce genome rearrangements if not accurately repaired. DSBs can be repaired either through homologous recombination (HR) or non-homologous end-joining (NHEJ). HR is the preferred repair pathway during the S and G2 cell cycle phases because a sister chromatid provides a perfect template for ‘error-free'' repair. During G1, when HR is suppressed to prevent recombination with homologues, repair is achieved primarily by NHEJ. Molecularly, DSB repair pathway choice is largely regulated at the level of 5′ to 3′ DNA end resection, that is, the formation of the 3′ end single-stranded DNA overhangs that are used to initiate HR. End resection inhibits NHEJ and promotes HR.In the June issue of Cell, Nussenzweig and colleagues identified the protein PTIP (also known as PAXIP) as a new component of the regulatory network that controls DSB repair pathway choice [1]. This work has important implications for our understanding of the mechanisms by which genomic integrity is underpinned, and is especially germane to those interested in the genesis of breast and ovarian cancer caused by a defective BRCA1 protein, which is crucial for DSB repair by HR.53BP1 (also known as TP53BP1) is a key determinant of DSB repair pathway choice [2]. In response to DSBs, 53BP1 binds to chromatin at damaged sites, where it promotes NHEJ by blocking end resection. 53BP1 has a crucial role during class switch recombination (CSR) in B cells and the fusion of dysfunctional telomeres. An even more striking phenotype was observed in mice in which loss of 53BP1 reversed most of the phenotypes associated with BRCA1 deficiency, including cell and embryonic lethality as well as tumorigenesis [2]. These findings suggest that 53BP1 and BRCA1 battle each other to influence DSB repair pathway choice.Molecularly, 53BP1 is responsible for the defective HR seen in BRCA1-deficient cells. Furthermore, in those cells, 53BP1 promotes the formation of characteristic radial chromosomes that are caused by toxic NHEJ events, presumably during S phase. Understanding exactly how 53BP1 carries out its many functions has been a major challenge to the field as 53BP1 does not harbour any enzymatic activity. However, it has been shown that 53BP1 must accumulate on chromatin to be functional. In addition, a mutant 53BP1 allele in which all 28 ataxia telangiectasia-mutated (ATM) phosphorylation sites were changed to alanine (53BP1^28A) failed to rescue 53BP1 deficiency, suggesting that 53BP1 acts through phosphorylation-dependent protein interactions to promote NHEJ [2].RIF1 was identified as the first effector of 53BP1 in DSB repair [3,4,5,6,7]. RIF1 accumulates at DSB sites by binding to phosphorylated 53BP1 but, intriguingly, the loss of RIF1 has a milder effect than the loss of 53BP1 with respect to the fusion of dysfunctional telomeres [3], and RIF1 deficiency does not fully restore HR in BRCA1-deficient cells [7]. As the 53BP1^28A mutant is nearly as defective as the complete loss of 53BP1 for these activities, these observations indicate that additional 53BP1 effector proteins contribute to some of the 53BP1 functions.Nussenzweig and colleagues provide compelling evidence that the BRCT domain-containing protein PTIP is the missing 53BP1 effector protein [1]. The authors identified a separation-of-function mutation in 53BP1 that disrupted the first eight amino-terminal ATM sites (53BP1^8A). The 53BP1^8A mutant behaved the same as the wild-type protein with respect to CSR—a physiological process dependent on NHEJ—but failed to promote genome instability (radial chromosome formation) in BRCA1-deficient cells after treatment with a PARP inhibitor. Since RIF1-deficient cells have impaired CSR and RIF1 can localize to break sites in cells expressing the 53BP1^8A mutant, this suggests that a protein other than RIF1 binds to the N-terminal region of 53BP1 to inhibit HR.The newly identified 53BP1 effector protein PTIP is a multifunctional DNA repair factor that interacts with phosphorylated Ser 25 of 53BP1 through its tandem BRCT domains [8]—a site that was mutated in the 53BP1^8A allele. PTIP is also part of the MLL3/MLL4 histone H3 Lys 4 methyltransferase complexes but this function seems to be unrelated to its role as a 53BP1 co-factor.Nussenzweig and co-workers found that PTIP-deficient cells are sensitive to ionizing radiation but tolerant of DNA damaging agents that are toxic to HR-deficient cells, which suggests a role for PTIP in NHEJ. In agreement with this, the fusion frequency of uncapped telomeres was reduced in PTIP-deficient cells. Interestingly, as in the case of the 53BP1^8A allele, PTIP-deficient B cells were proficient in switching their immunoglobulin locus, although this switching event is impaired in RIF1^−/− B cells. This suggests that PTIP might participate selectively in pathological NHEJ.Nussenzweig and colleagues next generated a conditional BRCA1^−/− PTIP^−/− mouse to investigate the contribution of PTIP to the genome instability of BRCA1-deficient B cells. Loss of PTIP restored normal growth kinetics and genome stability to BRCA1-deficient cells treated with a PARP inhibitor. In addition, RAD51 IR-induced focus formation was restored in BRCA1^−/− PTIP^−/− cells. As the primary defect of BRCA1-deficient cells with respect to HR seems to be at the level of resection, the accumulation of the single-stranded DNA-binding protein RPA into IR-induced foci was then analysed. The finding that PTIP-deficient cells have an increased number of RPA foci per cell supports a role for PTIP in blocking resection. Together, this suggests that PTIP opposes DNA end resection and mutagenic DSB repair in BRCA1-deficient cells.These results were surprising as they revealed that the 53BP1 activities relating to physiological NHEJ (during CSR) and mutagenic NHEJ (after PARP inhibition) can be separated, and that they are carried out by two distinct proteins that ‘read'' ATM-dependent 53BP1 phosphorylation. The relationship between 53BP1, RIF1 and PTIP is probably complex, as suggested by the possible competition between RIF1 and PTIP, and the observation that both proteins contribute in an additive manner to the fusion of dysfunctional telomeres, downstream from 53BP1.According to these findings, multiple phosphorylation events in 53BP1 seem to integrate ATM activity to control distinct aspects of DSB repair pathway choice (Fig 1). Establishing exactly how an increase of ATM activity at break sites is translated into the coordination of 53BP1 phosphorylation, with RIF1 and PTIP binding, will be an important milestone towards understanding 53BP1 function. Indeed, multi-site phosphorylation and its recognition by binding proteins can be used to develop switch-like responses that might be important for organizing the chromatin at DSB sites.Open in a separate windowFigure 153BP1 phospho-dependent interactions involved in DSB repair. PTIP and RIF1 interact with chromatin-bound and ATM-phosphorylated 53BP1 at DSB sites. PTIP binds directly to 53BP1 phosphorylated on Ser 14;25 (within the first eight Ser/Thr-Q sites). RIF1 binds to phosphorylated 53BP1 either directly or through an intermediate factor (X). The carboxy-terminal seven Ser/Thr-Q sites (9–15 Ser/Thr-Q sites) are involved in the interaction of RIF1–53BP1, although the amino-terminal eight Ser/Thr-Q sites might stabilize the binding. It is unknown whether PTIP and RIF1 can associate simultaneously with 53BP1 (left side of the figure), or if the binding is exclusive, due to either differential phosphorylation of the Ser/Thr-Q sites or steric hindrance (right side of the figure). 53BP1, PTIP and RIF1 block DNA end-resection and promote NHEJ repair. Although both PTIP and RIF1 contribute to dysfunctional telomere fusions, they also have distinct functions downstream from 53BP1. While RIF1 is essential for CSR and has a milder effect on toxic NHEJ events, PTIP is dispensable for CSR and has a more prominent role in toxic NHEJ events that lead to genome instability in BRCA1-deficient cells. ATM, ataxia telangiectasia-mutated; CSR, class switch recombination; DSB, double-stranded break; NHEJ, non-homologous end-joining.The identification of PTIP as a new 53BP1 effector also deepens the mystery of DSB repair pathway choice regulation by 53BP1. Future studies are needed to elucidate how 53BP1 and its effector proteins block resection. Are PTIP and RIF1 blocking specific nucleases? Do they act in a temporally distinct fashion or are they distributed in distinct subdomains of the chromatin flanking DSB sites? What is the function of PTIP in relation to the cell cycle? Testing whether RIF1 binds directly to 53BP1, and if so to which phosphorylated site, might answer some of the above questions. The identification of a RIF1 mutation that selectively disrupts 53BP1 binding would enable surgical manipulation of the 53BP1–RIF1–PTIP circuit at DSB sites.Another unresolved issue is whether 53BP1 acts solely by recruiting RIF1 and PTIP, or whether 53BP1 has a more active role in blocking resection. We have shown that 53BP1 localizes to the chromatin flanking the DSBs by binding to methylated and ubiquitinated nucleosomes, in a wheel clamp-like manner [9]. This suggests that 53BP1 might modify the nucleosomal array structure in a way that makes it refractory to the resection machinery. Recognizing how nucleosomes modified by 53BP1 cooperate with RIF1 and PTIP might provide clues to the role of these two proteins in end protection.It is important to note that in human cells, PTIP might not be recruited to DSB sites in a 53BP1- and ATM-dependent manner [8]. Furthermore, in the avian B-cell line DT40, PTIP promotes HR instead of inhibiting it [10]. It will be important to revisit these studies to tease out whether these differences are due to context-, experiment- or species-specific effects.The identification of PTIP as a candidate genetic modifier of BRCA1-deficient tumours is an important finding. As noted by the authors, disabling the PTIP–53BP1 interaction pharmacologically might selectively restore HR in BRCA1-deficient cells, which might be useful in certain contexts, for example as a chemopreventive strategy.     

Playing the end game: DNA double-strand break repair pathway choice

DNA double-strand breaks (DSBs) are highly toxic lesions that can drive genetic instability. To preserve genome integrity, organisms have evolved several DSB repair mechanisms, of which nonhomologous end-joining (NHEJ) and homologous recombination (HR) represent the two most prominent. It has recently become apparent that multiple layers of regulation exist to ensure these repair pathways are accurate and restricted to the appropriate cellular contexts. Such regulation is crucial, as failure to properly execute DSB repair is known to accelerate tumorigenesis and is associated with several human genetic syndromes. Here, we review recent insights into the mechanisms that influence the choice between competing DSB repair pathways, how this is regulated during the cell cycle, and how imbalances in this equilibrium result in genome instability.     

The fission yeast UVDR DNA repair pathway is inducible.

In addition to nucleotide excision repair (NER), the fission yeast Schizosaccharomyces pombe possesses a UV damage endonuclease (UVDE) for the excision of cyclobutane pyrimidine dimers and 6-4 pyrimidine pyrimidones. We have previously described UVDE as part of an alternative excision repair pathway, UVDR, for UV damage repair. The existence of two excision repair processes has long been postulated to exist in S.pombe, as NER-deficient mutants are still proficient in the excision of UV photoproducts. UVDE recognizes the phosphodiester bond immediately 5'of the UV photoproducts as the initiating event in this process. We show here that UVDE activity is inducible at both the level of uve1+ mRNA and UVDE enzyme activity. Further, we show that UVDE activity is regulated by the product of the rad12 gene.     

DNA damage response and cancer therapeutics through the lens of the Fanconi Anemia DNA repair pathway

Fanconi Anemia (FA) is a rare, inherited genomic instability disorder, caused by mutations in genes involved in the repair of interstrand DNA crosslinks (ICLs). The FA signaling network contains a unique nuclear protein complex that mediates the monoubiquitylation of the FANCD2 and FANCI heterodimer, and coordinates activities of the downstream DNA repair pathway including nucleotide excision repair, translesion synthesis, and homologous recombination. FA proteins act at different steps of ICL repair in sensing, recognition and processing of DNA lesions. The multi-protein network is tightly regulated by complex mechanisms, such as ubiquitination, phosphorylation, and degradation signals that are critical for the maintenance of genome integrity and suppressing tumorigenesis. Here, we discuss recent advances in our understanding of how the FA proteins participate in ICL repair and regulation of the FA signaling network that assures the safeguard of the genome. We further discuss the potential application of designing small molecule inhibitors that inhibit the FA pathway and are synthetic lethal with DNA repair enzymes that can be used for cancer therapeutics.     

ATM activates the pentose phosphate pathway promoting anti-oxidant defence and DNA repair

Ataxia telangiectasia (A-T) is a human disease caused by ATM deficiency characterized among other symptoms by radiosensitivity, cancer, sterility, immunodeficiency and neurological defects. ATM controls several aspects of cell cycle and promotes repair of double strand breaks (DSBs). This probably accounts for most of A-T clinical manifestations. However, an impaired response to reactive oxygen species (ROS) might also contribute to A-T pathogenesis. Here, we show that ATM promotes an anti-oxidant response by regulating the pentose phosphate pathway (PPP). ATM activation induces glucose-6-phosphate dehydrogenase (G6PD) activity, the limiting enzyme of the PPP responsible for the production of NADPH, an essential anti-oxidant cofactor. ATM promotes Hsp27 phosphorylation and binding to G6PD, stimulating its activity. We also show that ATM-dependent PPP stimulation increases nucleotide production and that G6PD-deficient cells are impaired for DSB repair. These data suggest that ATM protects cells from ROS accumulation by stimulating NADPH production and promoting the synthesis of nucleotides required for the repair of DSBs.     

A new approach to the study of the base-excision repair pathway using methoxyamine

This paper describes the use of methoxyamine to study the enzymatic reactions catalyzed by uracil-DNA glycosylase and by AP (apurinic/apyrimidinic) endodeoxyribonuclease isolated from mammalian cells. [14C]Methoxyamine permits one to follow the formation of AP sites in a uracil-containing polydeoxyribonucleotide incubated with calf thymus uracil-DNA glycosylase. The number of methoxyamine-reacted AP sites is equal to that of uracil released. Methoxyamine has no effect on the uracil-DNA glycosylase activity and may be added together with the enzyme in order to block the AP sites and prevent the degradation of the polynucleotide by the AP endonucleases that may be present in a crude preparation. Addition of methoxyamine to AP sites prevents not only the enzymatic hydrolysis of the adjacent phosphodiester bond but also the degradation of the polynucleotide by NaOH. This protective effect disappears after methoxyamine is removed by acetaldehyde.     

A Rad50-dependent pathway of DNA repair is deficient in Fanconi anemia fibroblasts

Fanconi anemia (FA) is a fatal genetic disorder associated with pancytopenia and cancer. Cells lacking functional FA genes are hypersensitive to bifunctional alkylating agents, and are deficient in DNA double-strand break repair. Multiple genes with FA-causing mutations have been cloned, however, the molecular basis for FA remains obscure. The results presented herein indicate that a Rad50-dependent end-joining process is non-functional in diploid fibroblasts from FA patients. Introduction of anti-Rad50 antibody into normal fibroblasts sensitized them to DNA damaging agents, whereas this treatment had no effect on fibroblasts from FA patients. The DNA end-joining process deficient in FA cells also requires the Mre11, Nbs1 and DNA ligase IV proteins. These data reveal the existence of a previously uncharacterized Rad50-dependent DNA double-strand break repair pathway in mammalian somatic cells, and suggest that failure to activate this pathway is responsible, at least in part, for the defective DNA end-joining observed in FA cells.     

Plant mitochondria possess a short-patch base excision DNA repair pathway

Despite constant threat of oxidative damage, sequence drift in mitochondrial and chloroplast DNA usually remains very low in plant species, indicating efficient defense and repair. Whereas the antioxidative defense in the different subcellular compartments is known, the information on DNA repair in plant organelles is still scarce. Focusing on the occurrence of uracil in the DNA, the present work demonstrates that plant mitochondria possess a base excision repair (BER) pathway. In vitro and in organello incision assays of double-stranded oligodeoxyribonucleotides showed that mitochondria isolated from plant cells contain DNA glycosylase activity specific for uracil cleavage. A major proportion of the uracil–DNA glycosylase (UDG) was associated with the membranes, in agreement with the current hypothesis that the DNA is replicated, proofread and repaired in inner membrane-bound nucleoids. Full repair, from uracil excision to thymidine insertion and religation, was obtained in organello following import of a uracil-containing DNA fragment into isolated plant mitochondria. Repair occurred through single nucleotide insertion, which points to short-patch BER. In vivo targeting and in vitro import of GFP fusions showed that the putative UDG encoded by the At3g18 630 locus might be the first enzyme of this mitochondrial pathway in Arabidopsis thaliana.     

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。