原核生物的NHEJ修复途径

DNA双链断裂(DSBs)是严重的DNA损伤形式之一,生物体对DSBs的修复可通过同源重组(HR)或非同源末端连接途径(NHEJ)进行。长期以来,人们普遍认为HR是细菌DSBs修复的惟一途径,但在分支杆菌和其它原核生物体内NHEJ途径的发现,使这一观念得以颠覆。最近的研究表明,细菌NHEJ修复系统是一个双组分系统,包含一个多功能的DNA连接酶(LigD)和DNA末端结合蛋白Ku,具有DSBs修复所需的断裂末段识别、末端加工和连接活性。重点综述细菌NHEJ修复系统的组成、结构以及生理功能。     

Differences in repair profiles of interstitial telomeric sites between normal and DNA double-strand break repair deficient Chinese hamster cells

Interstitial Telomeric Repeat Sequence (ITRS) blocks are recognized as hot spots for spontaneous and ionizing radiation-induced chromosome breakage and recombination. Background and ionizing radiation-induced DNA breaks in large blocks of ITRS from Chinese hamster cell lines were analyzed using the DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) procedure. Our results indicate an extremely alkali-sensitivity of ITRS. Furthermore, it appears that ITRS blocks exhibit a particular chromatin structure, being enriched in short unpaired DNA segments. These segments could be liable to severe topological stress in highly compacted areas of the genome resulting in their spontaneous fragility and thus explaining their alkali-sensitivity. The induction and repair kinetics of DNA single-strand breaks (ssb) and DNA double-strand breaks (dsb) induced by ionizing radiation were assessed by DBD-FISH on neutral comets using Chinese hamster cells deficient in either DNA-PKcs or Rad51C. Our results indicate that the initial rejoining rate of dsb within ITRS is slower than that in the whole genome, in wild-type cells, demonstrating an intragenomic heterogeneity in dsb repair. Interestingly, in the absence of DNA-PKcs activity, the rejoining rate of dsb within ITRS is not modified, unlike in the whole genome. This was also found in the case of Rad51C mutant cells. Our results suggest the possibility that different DNA sequences or chromatin organizations may be targeted by specific dsb repair pathways. Furthermore, it appears that additional unknown dsb repair pathways may be operational in mammalian cells.     

Nanoscale analysis of clustered DNA damage after high-LET irradiation by quantitative electron microscopy – The heavy burden to repair

Low- and high-linear energy transfer (LET) ionising radiation are effective cancer therapies, but produce structurally different forms of DNA damage. Isolated DNA damage is repaired efficiently; however, clustered lesions may be more difficult to repair, and are considered as significant biological endpoints. We investigated the formation and repair of DNA double-strand breaks (DSBs) and clustered lesions in human fibroblasts after exposure to sparsely (low-LET; delivered by photons) and densely (high-LET; delivered by carbon ions) ionising radiation. DNA repair factors (pKu70, 53BP1, γH2AX, and pXRCC1) were detected using immunogold-labelling and electron microscopy, and spatiotemporal DNA damage patterns were analysed within the nuclear ultrastructure at the nanoscale level. By labelling activated Ku-heterodimers (pKu70) the number of DSBs was determined in electron-lucent euchromatin and electron-dense heterochromatin. Directly after low-LET exposure (5 min post-irradiation), single pKu70 dimers, which reflect isolated DSBs, were randomly distributed throughout the entire nucleus with a linear dose correlation up to 30 Gy. Most euchromatic DSBs were sensed and repaired within 40 min, whereas heterochromatic DSBs were processed with slower kinetics. Essentially all DNA lesions induced by low-LET irradiation were efficiently rejoined within 24 h post-irradiation. High-LET irradiation caused localised energy deposition within the particle tracks, and generated highly clustered DNA lesions with multiple DSBs in close proximity. The dimensions of these clustered lesions along the particle trajectories depended on the chromatin packing density, with huge DSB clusters predominantly localised in condensed heterochromatin. High-LET irradiation-induced clearly higher DSB yields than low-LET irradiation, with up to ∼500 DSBs per μm³ track volume, and large fractions of these heterochromatic DSBs remained unrepaired. Hence, the spacing and quantity of DSBs in clustered lesions influence DNA repair efficiency, and may determine the radiobiological outcome.     

聚腺苷二磷酸-核糖聚合酶1与DNA双链断裂修复的相关性

DNA双链断裂(double strand breaks,DSBs)是细胞最严重的DNA损伤形式。细胞通过同源重组(homologous recombination,HR)和非同源末端连接(non-homologous end joining,NHEJ)途径修复DNA双链断裂损伤。聚腺苷二磷酸核糖基化(poly(ADP-ribosyl)ation,PARylation)是蛋白质翻译后修饰过程,这个过程由聚腺苷二磷酸 核糖聚合酶家族(poly(ADP-ribose)polymerases,PARPs)催化完成。PARP1作为PARPs家族最重要的成员,其在DNA损伤应答方面发挥重要作用。研究显示,PARP1在DSBs修复过程中发挥关键作用,参与DSBs的早期应答反应及其具体修复途径,可依据KU蛋白的存在与否发挥不同的特定作用。本文较全面地综述了PARP1在DNA双链断裂修复方面的潜在作用,将为临床疾病的诊治提供新的思路。     

XRCC4 controls nuclear import and distribution of Ligase IV and exchanges faster at damaged DNA in complex with Ligase IV

Non-homologous end-joining (NHEJ) is one major pathway for the repair of double-stranded DNA breaks in mammals. Following break recognition, alignment and processing, broken DNA ends are finally rejoined by the essential DNA Ligase IV. In the cell, Ligase IV is unable to function without its constitutive interaction partner XRCC4 and becomes unstable when it is missing, and it has been assumed that XRCC4 may also be required for recruitment of Ligase IV to repair sites. To investigate the function of complex formation between both proteins directly in the living cell, we stably expressed them as bio-fluorescent fusion proteins in human HT-1080 cell clones. Ligase IV or XRCC4 were expressed either alone or both were co-expressed at a roughly equimolar ratio. Labelled proteins were overexpressed manifold in comparison to endogenously expressed proteins. We show that over-expressed Ligase IV was only partially imported into the nucleus and showed a diffuse distribution there, whereas XRCC4 expressed alone was entirely nuclear with a distinct exclusion from nucleoli. When Ligase IV was co-expressed with XRCC4, both proteins formed the natural complex, and Ligase IV was not only efficiently imported but also resembled the sub-nuclear distribution of XRCC4. In addition, Ligase IV, when in complex with XRCC4, acquired a delayed nuclear reimport after mitotic cell division of XRCC4. We further determined by photobleaching the kinetics with which the proteins exchange at UVA laser-irradiated nuclear sites between damage-bound and diffusing states. We found that the dynamic exchange rate of the Ligase IV/XRCC4 complex at micro-irradiated sites was faster than that of XRCC4 expressed alone. In summary, our findings demonstrate a novel function of XRCC4 in controlling nuclear import and sub-nuclear distribution of Ligase IV, and they suggest that XRCC4 modulates the dynamic interaction of the Ligase IV/XRCC4 complex with the NHEJ machinery at double-stranded DNA breaks.     

Non-homologous DNA end joining in the mature rat brain

Recent evidence suggests that DNA double strand breaks (DSBs) are introduced in neurons during the course of normal development, and that repair of such DSBs is essential for neuronal survival. Here we describe a non-homologous DNA end joining (NHEJ) system in the adult rat brain that may be used to repair DNA DSBs. In the brain NHEJ system, blunt DNA ends are joined with lower efficiency than cohesive or non-matching protruding ends. Moreover, brain NHEJ is blocked by DNA ligase inhibitors or by dATP and can occur in the presence or absence of exogenously added ATP. Comparison of NHEJ activities in several tissues showed that brain and testis share similar mechanisms for DNA end joining, whereas the activity in thymus seems to utilize different mechanisms than in the nervous system. The developmental profile of brain NHEJ showed increasing levels of activity after birth, peaking at postnatal day 12 and then gradually decreasing along with age. Brain distribution analysis in adult animals showed that NHEJ activity is differentially distributed among different regions. We suggest that the DNA NHEJ system may be utilized in the postnatal brain for the repair of DNA double strand breaks introduced within the genome in the postnatal brain.     

Accumulation of DNA damage in complex normal tissues after protracted low-dose radiation

The biological consequences of low levels of radiation exposure and their effects on human health are unclear. Ionizing radiation induces a variety of lesions of which DNA double-strand breaks (DSBs) are the most biologically significant, because unrepaired or misrepaired DSBs can lead to genomic instability and cell death. Using repair-proficient mice as an in vivo system we monitored the accumulation of DNA damage in normal tissues exposed to daily low-dose radiation of 100 mGy or 10 mGy. Radiation-induced foci in differentiated and tissue-specific stem cells were quantified by immunofluorescence microscopy after 2, 4, 6, 8, and 10 weeks of daily low-dose radiation and DNA lesions were characterized using transmission electron microscopy (TEM) combined with immunogold-labeling. In brain, long-living cortical neurons had a significant accumulation of foci with increasing cumulative doses. In intestine and skin, characterized by constant cell renewal of their epithelial lining, differentiated enterocytes and keratinocytes had either unchanged or only slightly increased foci levels during protracted low-dose radiation. Significantly, analysis of epidermal stem cells in skin revealed a constant increase of 53BP1 foci during the first weeks of low-dose radiation even with 10 mGy, suggesting substantial accumulations of DSBs. However, TEM analysis suggests that these remaining 53BP1 foci, which are predominantly located in compact heterochromatin, do not co-localize with phosphorylated Ku70 or DNA-PKcs, core components of non-homologous end-joining. The biological relevance of these persistent 53BP1 foci, particularly their contribution to genomic instability by genetic and epigenetic alterations, has to be defined in future studies.     

Backup pathways of NHEJ are suppressed by DNA-PK

In cells of higher eukaryotes double strand breaks (DSBs) induced in the DNA after exposure to ionizing radiation (IR) are rapidly rejoined by a pathway of non-homologous end joining (NHEJ) that requires DNA dependent protein kinase (DNA-PK) and is therefore termed here D-NHEJ. When this pathway is chemically or genetically inactivated, cells still remove the majority of DSBs using an alternative, backup pathway operating independently of the RAD52 epistasis group of genes and with an order of magnitude slower kinetics (B-NHEJ). Here, we investigate the role of DNA-PK in the functional coordination of D-NHEJ and B-NHEJ using as a model end joining by cell extracts of restriction endonuclease linearized plasmid DNA. Although DNA end joining is inhibited by wortmannin, an inhibitor of DNA-PK, the degree of inhibition depends on the ratio between DNA ends and DNA-PK, suggesting that binding of inactive DNA-PK to DNA ends not only blocks processing by D-NHEJ, but also prevents the function of B-NHEJ. Residual end joining under conditions of incomplete inhibition, or in cells lacking DNA-PK, is attributed to the function of B-NHEJ operating on DNA ends free of DNA-PK. Thus, DNA-PK suppresses alternative pathways of end joining by efficiently binding DNA ends and shunting them to D-NHEJ.     

Non-homologous end joining plays a key role in transgene concatemer formation in transgenic zebrafish embryos

This study focused on concatemer formation and integration pattern of transgenes in zebrafish embryos. A reporter plasmid based on enhanced green fluorescent protein (eGFP) driven by Cytomegalovirus (CMV) promoter, pCMV-pax6in-eGFP, was constructed to reflect transgene behavior in the host environment. After removal of the insertion fragment by double digestion with various combinations of restriction enzymes, linearized pCMV-pax6in-eGFP vectors were generated with different combinations of 5'-protruding, 3'-protruding, and blunt ends that were microinjected into zebrafish embryos. Repair of double-strand breaks (DSBs) was monitored by GFP expression following religation of the reporter gene. One-hundred-and-ninety-seven DNA fragments were amplified from GFP-positive embryos and sequenced to analyze the repair characteristics of different DSB end combinations. DSBs involving blunt and asymmetric protruding ends were repaired efficiently by direct ligation of blunt ends, ligation after blunting and fill-in, or removed by cutting. Repair of DSBs with symmetric 3'-3' protrusions was less efficient and utilized template-directed repair. The results suggest that non-homologous end joining (NHEJ) was the principal mechanism of exogenous gene concatemer formation and integration of transgenes into the genome of transgenic zebrafish.     

DNA双链断裂损伤修复系统研究进展

多种内源或外源因素都能造成细胞基因组DNA损伤,细胞内建立了复杂的修复系统来应对不同形式的损伤。其中DNA双链断裂(DNA double-strand breaks,DSBs)作为最严重的损伤形式,主要激活同源重组修复(Homologous recombination repair)和非同源末端连接(Non-homologous end joining)通路。这两条通路都是由多个修复元件参与、经过多步反应的复杂过程。两者各具特点、协同作用,共同维护细胞基因组的稳定性。对其分子机制的阐明为肿瘤放化疗的辅助治疗提供了潜在的作用靶点。