正常人淋巴细胞程序外DNA合成的水平

程序外DNA合成(unscheduled DNA synthesis,UDS)反映了细胞对DNA损伤进行切除修复的能力,在毒理学、肿瘤学、放射医学等多种学科中都有应用。关于我国正常人外周血淋巴细胞UDS的水平,仅在近几年才有零星的附带观察,例数都较少,而专文报道,迄今未见。本文报告对60例正常人的观察结果。 1 材料与方法1.1 血液样品来源 本院医护人员(除外放射科     

抗氧化剂对脂质过氧化反应混合物引起人培养细胞DNA损伤的影响

 <正> 脂质过氧化反应(LPR)中产生的自由基和过氧化物可引起多种细胞损伤,特别是对DNA的损伤可能是许多癌肿的重要原因。Akasaka等将E.coli与鼠肝微粒体脂质过氧化反应混合物(LPRM)孵育,使有DNA修复缺陷的细菌株突变率增加。我们将人羊膜FL细胞与大鼠肝微粒体LPRM共同培养,看能否诱发程序外DNA合成(UDS),并分组加入抗氧化剂观察其影响。     

DNA损伤修复基本方式的研究进展

DNA损伤修复基因可修复由不同原因导致的DNA损伤．从而保护遗传信息的完整性。DNA损伤修复有3种基本形式,即碱基切除修复、核苷酸切除修复和错配修复。本文综述了DNA损伤修复3种基本形式的研究进展情况并讨论了DNA链断裂重组和重接合修复及DNA聚合酶绕道修复DNA损伤。     

自噬与DNA损伤修复及其对肿瘤的影响

DNA损伤与肿瘤的发生发展密切相关。当DNA损伤发生时,会触发一系列的损伤应答反应以帮助细胞生存,其中即包括对自噬的诱导。ATM、P53和PARP1等多种参与DNA损伤修复的效应因子通过影响AMPK、mTOR以及一些凋亡蛋白等启动自噬。而作为一种降解途径,自噬则可通过调节DNA修复相关蛋白的水平直接影响同源重组修复、非同源末端连接修复和核苷酸切除修复等促进DNA修复,以及通过维持细胞内稳态间接促进DNA修复,从而在正常细胞的恶性转化和肿瘤耐药等发生机制中扮演重要角色。此外,DNA修复失败时,自噬也可作为一种肿瘤细胞的程序性死亡方式。因此研究自噬通过调节DNA损伤修复而对肿瘤的影响对于理解肿瘤发生的机制和提供治疗思路都有重要意义。     

紫外线诱导DNA损伤及修复研究的起因与发展

人类在DNA修复研究方面的重大突破主要来自紫外线诱导DNA损伤及修复研究,而该研究对于人类了解基因突变的机制、衰老和癌变的原因,以及应用于环境致癌因子的检测等方面具有重要意义。对以光修复为主的DNA直接修复和具有核苷酸切除修复、碱基切除修复、SOS修复3种基本形式的DNA切除修复的起因与发展历程进行综述。     

DNA损伤修复机制——解读2015年诺贝尔化学奖

Tomas Lindahl, Paul Modrich和Aziz Sancar三位科学家因发现“DNA损伤修复机制”获得了2015年诺贝尔化学奖．Lindahl首次发现Escherichia Coli中参与碱基切除修复的第一个蛋白质--尿嘧啶 DNA糖基化酶（UNG）; Modrich重建了错配修复的体外系统,从大肠杆菌到哺乳动物深入探究了错配修复的机制; Sancar利用纯化的UvrA、UvrB、UvrC重建了核苷酸切除修复的关键步骤,阐述了核苷酸切除修复的分子机制．DNA损伤是由生物所处体外环境和体内因素共同导致的,面对不同种类的损伤,机体启动多种不同的修复机制修复损伤,保护基因组稳定性．这些修复机制包括：光修复(light repairing);核苷酸切除修复（nucleotide excision repair, NER）;碱基切除修复（base excision repair, BER）;错配修复（mismatch repair, MMR）;以及DNA双链断裂修复（DNA double strand breaks repair, DSBR）．其中DNA双链断裂修复又分同源重组（homologous recombination, HR）和非同源末端连接（non homologous end joining, NHEJ）两种方式．本文将对上述几种修复的机制进行总结与讨论．     

碱基切除修复在阿尔茨海默病和帕金森病中的作用

细胞代谢或细胞应激均可以引起DNA氧化损伤。DNA氧化损伤与神经退行性疾病的发生、发展密切相关。碱基切除修复在抵抗脑细胞DNA氧化损伤中起着重要的作用。就碱基切除修复在阿尔茨海默病(Alzheimer’s disease,AD)和帕金森病(Parkinson’s disease,PD)中的作用及其机制进行综述。     

螺旋藻多糖的生物活性研究概况

近两年对螺旋藻 (Ｓｐｉｒｕｌｉｎａ)的研究多集中在螺旋藻多糖上 ,根据所在位置不同而存在胞内多糖、囊鞘多糖和释放多糖。国内研究多集中在胞内多糖上 ,而国外对蓝绿藻的多糖研究集中在囊鞘多糖和释放多糖上。螺旋藻多糖因其参加了细胞的多种生命现象的调节作用 ,具有抗肿瘤、抗辐射、ＤＮＡ修复及免疫增强作用 ,成为国内外海洋药物研究的重点。本文综述国内外螺旋藻多糖生物活性的研究进展以及研究方法。1　螺旋藻多糖的免疫调节和延缓衰老功能左绍远等[1 ] 实验发现 ,小鼠ｉｐＰＳＰ1 0 0ｍｇ ｋｇ ,连续 77ｄ ,可明显增加脾重 ;显著促…     

DNA切除修复与转录偶联

细胞DNA受到某些环境理化因子损伤后,其中活性转录基因和DNA转录链上的损伤被优先切除修复,这种DNA选择性修复直接与基因转录过程偶联．在大肠杆菌中已分离到实现此功能的转录修复偶联因子（TRCF）,是由mdf基因编码的一种具有ATPase活性的DNA结合蛋白．在真核细胞中,发现某些DNA修复蛋白也在DNA转录中起作用,如人DNA切除修复基因ERCG－3编码产物,是转录因子TFⅡH中最大亚基p89,酵母切除修复基因RAD3就是编码因子b的最大亚基p85．     

DNA损伤修复蛋白在炎症免疫反应中的作用

细胞代谢或细胞应激均可以引起DNA损伤。DNA损伤可以引起一系列级联反应即DNA损伤反应。炎症免疫反应是活体组织对损伤因子所起的防御反应。DNA损伤反应与炎症的发生发展密切相关,而DNA损伤修复蛋白在免疫系统中具有重要作用。本文将就DNA损伤修复蛋白在炎症免疫反应中的作用及其机制进行综述。     

Mutations at the mei-41, mus(1)101, mus(1)103, mus(2)205 and mus(3)310 loci of Drosophila exhibit differential UDS responses with different DNA-damaging agents

5 mutagen-sensitive mutants of Drosophila melanogaster, reported to perform normal or only slightly reduced excision repair of UV damage, were examined by an unscheduled DNA synthesis (UDS) assay. This assay measures the ability of cultured primary cells, derived from each mutant, to perform the resynthesis step in the excision repair pathway, following damage to cellular DNA by direct-acting alkylating agents, UV or X-irradiation. 2 mutants, classified as completely or partially proficient for both excision and postreplication repair of UV damage, mus(1)103 and mus(2)205, were found to give positive UDS responses only for UV damage. These mutants exhibit no measurable UDS activity following DNA damage by several different alkylating agents and X-rays. 3 mutants, classified as having no defect in excision repair, but measurable defects in postreplication repair of UV damage, mei-41, mus(1)101, and mus(3)310 exhibit 3 different response patterns when tested with the battery of agents in the UDS assay. The mutant mei-41 exhibits a highly positive UDS response following damage by all agents, consistent with its prior classification as excision-repair-proficient, but postreplication-repair-deficient for UV damage. The mutant mus(1)101, however, exhibits a strong positive UDS response following only UV damage and appears to be blocked in the excision repair of damage produced by both alkylating agents and X-irradiation. Finally, mus(3)310 exhibits no UDS response to alkylation, X-ray or UV damage. This is not consistent with its previous classification. Results obtained with the quantitative in vitro UDS assay are entirely consistent with the results from two separate in vivo measures of excision repair deficiency following DNA damage, larval hypersensitivity to killing and hypermutability in the sex-linked recessive lethal test.     

Phenotypic heterogeneity within the first complementation group of UV-sensitive mutants of Chinese hamster cell lines

A DNA-repair mutant was characterized that has the extraordinary and interesting properties of extreme sensitivity to UV killing combined with a high level of nucleotide excision repair. The mutant V-H1 isolated from the V79 Chinese hamster cell line appeared very stable, with a reversion frequency of about 3.5 × 10⁻⁷. Genetic complementation analysis indicates that V-H1 belongs to the first complementation group of UV-sensitive Chinese hamster ovary (CHO) mutants described by Thompson et al. (1981). This correponds with data on cross-sensitivity and mutation induction after UV irradiation published by this group. Surprisingly, the mutant V-H1 shows only slightly reduced (to ∼ 70%) unscheduled DNA synthesis (UDS) after UV exposure, while the other two mutants of this complementation group are deficient in UDS after UV. In agreement with the high residual UDS, in V-H1 also the amount of repair replication in response to UV treatment is relatively high (∼ 50%). It has also been shown that the incision step of the nucleotide excision pathway takes place in V-H1 (with a lower rate than observed in wild-type cells), whereas another mutant (UV5) of the same complementation group is deficient in incision.This heterogeneity within the first complementation group indicates that the repair gene of this complementation group may have more than one functionally domain or that the gene is not involved in the incision per se but is involved in e.g. preferential repair of active genes.     

Nitropyrene-induced DNA repair in Clara cells and alveolar type-II cells isolated from rabbit lung

DNA excision repair, as measured by unscheduled DNA synthesis (UDS), was examined in different cell types of rabbit lung exposed to nitropolycyclic aromatic hydrocarbons (NO-PAH) in vitro. Dose-related increases in UDS were observed. 1,6-Dinitropyrene (1,6-DNP) and 1,8-dinitropyrene (1,8-DNP) induced UDS more effectively in alveolar type-II cells compared with Clara cells. On the other hand, 1-nitropyrene (1-NP) caused a weak UDS response in Clara cells but no DNA repair in alveolar type-II cells.     

A new flow cytometric method to follow DNA gap filling during nucleotide excision repair of UVc-induced damage

BACKGROUND: Several methods have been developed for studying the kinetics of DNA repair after exposure of cells to ultraviolet (UV) light, such as conventional assays measuring unscheduled DNA synthesis (UDS). In this study, we have developed an accurate and rapid method to follow DNA gap filling during nucleotide excision repair (NER) in normal human fibroblasts (NHFs) in response to UV-induced damage. METHODS: After UVc irradiation, aphidicolin was added to the culture to hold repair patches open. This allowed an efficient incorporation of biotin-21-dUTP during an endogenous DNA repair synthesis that was detected by flow cytometry. RESULTS: We showed that the DNA gap filling after UVc irradiation in NHFs increased with time up to 10 h after irradiation and that no repair synthesis activity could be detected in XP-A fibroblasts. Furthermore, this activity was UVc dose dependent up to 20 J/m2. These results correlated well with those of the UDS assay. Interestingly, addition of aphidicolin at different time points after UVc irradiation, thus allowing endogenous repair synthesis in the absence of biotin-21-dUTP, demonstrated that the response of the NER system occurred extremely rapidly after irradiation. CONCLUSIONS: This method may be a reliable and simple alternative to other techniques measuring UDS. Practical advantages include the rapidity of the method, no need for radioactivity, and the possibility to use a second and/even a third flow marker to analyse cell cycle and heterogeneous cell populations concomitantly.     

Drosophila mutations at the mei-9 and mus(2)201 loci which block excision of thymine dimers also block induction of unscheduled DNA synthesis by methyl methanesulfonate, ethyl methanesulfonate, N-methyl-N-nitrosourea, UV light and X-rays

The mei-9 and mus(2)201 mutants of Drosophila melanogaster were identified as mutagen-sensitive mutants on the basis of larval hypersensitivity to methyl methanesulfonate and characterized as excision repair-deficient on the basis of a greatly reduced capacity to excise thymine dimers from cellular DNA. The high degree of larval cytotoxicity observed with a variety of other chemical and physical agents indicated that these mutants may be unable to excise other important classes of DNA adducts. We have measured the ability of the single mutants and the double mutant combination mei-9;mus(2)201 to perform the resynthesis step in excision repair by means of an autoradiographic analysis of unscheduled DNA synthesis (UDS) induced in a mixed population of primary cells in culture. The 3 strains exhibit no detectable UDS activity in response to applied doses of 1.5-6.0 mM methyl methanesulfonate, 1.0-4.5 mM N-methyl-N-nitrosourea or 10-40 J/m2 254-nm UV light, dose ranges in which control cells exhibit a strong dose-dependent UDS response. The mei-9 and mei-9;mus(2)201 mutants also have no detectable UDS response to X-ray doses of 300-1800 rad, whereas the mus(2)201 mutant exhibits a reduced, but dose-dependent, response over this range. These data correlate well with the degree of larval hypersensitivity of the strains and suggest that mutations at both loci block the excision repair of a wide variety of DNA damage prior to the resynthesis step.     

Human nucleotide excision repair in vitro: repair of pyrimidine dimers, psoralen and cisplatin adducts by HeLa cell-free extract.

We searched for nucleotide excision repair in human cell-free extracts using two assays: damage-specific incision of DNA (the nicking assay) and damage-stimulated DNA synthesis (the repair synthesis assay). HeLa cell-free extract prepared by the method of Manley et al. (1980) has a weak nicking activity on UV irradiated DNA and the nicking is only slightly reduced when pyrimidine dimers are eliminated from the substrate by DNA photolyase. In contrast to the nicking assay, the extract gives a strong signal with UV irradiated substrate in the repair synthesis assay. The repair synthesis activity is ATP dependent and is reduced by about 50% by prior treatment of the substrate with DNA photolyase indicating that this fraction of repair synthesis is due to removal of pyrimidine dimers by nucleotide excision. Psoralen and cisplatin adducts which are known to be removed by nucleotide excision repair also elicited repair synthesis activity 5-10 fold above the background synthesis. When M13RF DNA containing a uniquely placed psoralen adduct was used in the reaction, complete repair was achieved in a fraction of molecules as evidenced by the restoration of psoralen inactivated KpnI restriction site. This activity is absent in xeroderma pigmentosum group A cells. We conclude that our cell-free extract contains the human nucleotide excision repair enzyme activity.     

Two types of proliferating cell nuclear antigen (PCNA) complex formation in quiescent normal and xeroderma pigmentosum group A fibroblasts following ultraviolet light (uv) irradiation.

We examined the relationship between the formation of proliferating cell nuclear antigen (PCNA) complex with DNA and nucleotide excision repair in human fibroblasts following ultraviolet light (uv) irradiation. PCNA complex formation was detected by the immunofluorescence method after methanol fixation and nucleotide excision repair activity was detected as the unscheduled DNA synthesis (UDS) by autoradiography labeled with [3H]thymidine. Quiescent normal cells showed a strong punctuated pattern of PCNA staining 5 min to 3 h and UDS 3 h after 10 J/m2 of uv irradiation, but they no longer showed PCNA staining and UDS 24 h after irradiation. In contrast, xeroderma pigmentosum group A (XP-A) cells, which lack UDS activity, did not show PCNA staining up to 30 min after irradiation; however, unexpectedly, they were stained 3 h and even 24 h after irradiation with their staining pattern being different from that in normal cells. Namely, the fluorescence spots in XP-A cells were larger in size and much smaller in number than those in normal cells. When XP-A cells were fused with normal cells with polyethylene glycol treatment, nuclei of XP-A cells showed a PCNA staining pattern similar to that of normal cells at 30 min, which was no longer detected 24 h after irradiation. These results suggest that there exist two types of PCNA complex formation, nucleotide excision repair-related and -unrelated, in human fibroblasts following uv irradiation.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

UV-induced DNA excision repair in rat fibroblasts during immortalization and terminal differentiation in vitro

UV-induced DNA excision repair was studied as DNA repair synthesis and dimer removal in rat fibroblast cultures, initiated from either dense or sparse inocula of primary cells grown from skin biopsies. During passaging in vitro an initial increase in DNA repair synthesis, determined both autoradiographically as unscheduled DNA synthesis (UDS) and by means of the BrdU photolysis assay as the number and average size of repair patches, was found to be associated with a morphological shift from small spindle-shaped to large pleiomorphic cells observed over the first twenty generations. In cell populations in growth crisis, a situation exclusively associated with thin-inoculum cultures in which the population predominantly consisted of large pleiomorphic cells, UDS was found to occur at a low level. After development of secondary cultures into immortal cell lines, both repair synthesis and morphology appeared to be the same as in the original primary spindle-shaped cells. At all passages the capacity to remove UV-induced pyrimidine dimers was found to be low, as indicated by the persistence of Micrococcus luteus UV endonuclease-sensitive sites. These results are discussed in the context of terminal differentiation and immortalization of rat fibroblasts upon establishment in vitro.     

Repair of damage by ultraviolet radiation in xeroderma pigmentosum cell strains of complementation groups E and F

The xeroderma pigmentosum fibroblast strains XP2RO, complementation group E, and XP23OS, group F, were compared with normal human primary fibroblasts with regard to repair of damage induced by 254-nm UV. In XP2RO cells, repair DNA synthesis, measured by autoradiography (unscheduled DNA synthesis = UDS), was about 50% of the value found in normal human cells. In these cells also the removal of UV-induced sites recognized by a specific UV-endonuclease proceeds at a reduced rate. By having BUdR incorporated into the repaired regions, followed by the induction of breaks in these patches by 313-nm UV, it was shown that the reduced repair synthesis is not caused by a shorter length of the repair regions in XP2RO, but is solely due to a reduction in the number of sites removed by excision repair. In XP23OS a discrepancy was observed between the level of UDS, which was about 10% of the normal value, and other repair-dependent properties such as UV survival, host-cell reactivation and removal of UV-endonuclease-susceptible sites, which were less reduced than could be expected from the UDS level. However, when UDS was followed over a longer period than the 2 or 3 h normally used in UDS analysis, it appeared that in XP23OS cells, the rate of UDS remained constant whereas the rate decreased in normal control cells. Consequently, the residual level of UDS varies with the period over which it is studied.