多-ADP-核糖基化和DNA切除修复的关系

早在三十年前就发现某些化学物质或电离辐射造成DNA损伤时,细胞内NAD~+含量减少.现在证明,NAD~+除作为脱氢酶的辅酶外,还作为多-ADP-核糖合成酶的底物,用以合成多-ADP-核糖,从而修饰核蛋白发挥生物效应,如参与DNA的复制及修复等.多-ADP-核糖合成酶的活性是DNA依赖的,而且和DNA链上的切口数有关,但多-ADP-核糖基化和DNA修复过程中的哪些反应步骤有关,目前尚无定论. 本文以人淋巴细胞为材料,紫外线(UV)照射造成DNA损伤,研究多-ADP-核糖基化     

多-ADP-核糖基化和DNA切除修复的关系

早在三十年前就发现某些化学物质或电离辐射造成DNA损伤时,细胞内NAD~ 含量减少。现在证明,NAD~ 除作为脱氢酶的辅酶外,还作为多-ADP-核糖合成酶的底物,用以合成多-ADP-核糖,从而修饰核蛋白发挥生物效应,如参与DNA的复制及修复等。多-ADP-核糖合成酶的活性是DNA依赖的,而且和DNA链上的切口数有关,但多-ADP-核糖基化和DNA修复过程中的哪些反应步骤有关,目前尚无定论。本文以人淋巴细胞为材料,紫外线(UV)照射造成DNA损伤,研究多-ADP-核糖基化     

~(60)Co γ线对肿瘤病人T、B淋巴细胞转化过程中三种大分子合成的效应

应用植物血凝素(PHA)和脂多糖(LPS)激活淋巴细胞,以氢-胸腺嘧啶核苷(~3H-TdR)、碳一尿嘧啶核苷(~(14)C-UR)和碳-缬氨酸作掺入实验,以分别反映T、B淋巴细胞转化过程中的DNA、RNA和蛋白质的合成能力。共测定了50例肿瘤病人,与正常人比较:T淋巴细胞转化能力明显降低,B淋巴细胞转化能力显著增高。经过一个疗程的~(60)Co照射后T细胞转化比照前又显著降低,B细胞转化比照前又显著增高,三种大分子合成都表现同样的规律,反映了辐射抑制T淋巴细胞DNA和RNA合成,相反地刺激了B淋巴细胞的DNA、RNA和蛋白质的合成。以上的辐射效应随照射剂量和照射野的增加而愈益明显。     

Sulfolobus acidocaldarius DNA聚合酶IV跨越损伤合成能力的酶学特征

[目的]以嗜酸嗜热硫化叶菌Sulfolobus acidocaldarius的DNA聚合酶IV （Saci_0554）为例,表征其跨越模板上损伤碱基的DNA合成效果。[方法]将DNA聚合酶IV （SacpolIV）在大肠杆菌中进行重组表达,经亲和层析纯化得到SacpolIV蛋白;利用人工合成的带有不同损伤的寡核苷酸片段作为模板DNA,用尿素变性聚丙烯酰胺凝胶电泳技术,鉴定SacpolIV在体外跨越各种损伤碱基进行跨损伤合成的催化能力。[结果]SacpolIV重组蛋白能够不同程度地跨越嘌呤和嘧啶损伤,跨越能力的高低取决于损伤碱基与正常碱基形成氢键的能力。本研究还发现,SacpolIV能够在DNA链中掺入核糖核苷酸,但掺入核糖核苷酸的效率低于脱氧核糖核苷酸。[结论]本研究证实SacpolIV具有很强的跨越损伤合成能力,能够跨越多种氢键配对能力减弱的损伤碱基,为其在细胞内的跨越损伤合成功能提供了生化证据。     

Sulfolobus acidocaldarius DNA聚合酶Ⅳ跨越损伤合成能力的酶学特征

【目的】以嗜酸嗜热硫化叶菌Sulfolobus acidocaldarius的DNA聚合酶Ⅳ (Saci_0554)为例,表征其跨越模板上损伤碱基的DNA合成效果。【方法】将DNA聚合酶Ⅳ (SacpolⅣ)在大肠杆菌中进行重组表达,经亲和层析纯化得到SacpolⅣ蛋白;利用人工合成的带有不同损伤的寡核苷酸片段作为模板DNA,用尿素变性聚丙烯酰胺凝胶电泳技术,鉴定SacpolⅣ在体外跨越各种损伤碱基进行跨损伤合成的催化能力。【结果】SacpolⅣ重组蛋白能够不同程度地跨越嘌呤和嘧啶损伤,跨越能力的高低取决于损伤碱基与正常碱基形成氢键的能力。本研究还发现,SacpolⅣ能够在DNA链中掺入核糖核苷酸,但掺入核糖核苷酸的效率低于脱氧核糖核苷酸。【结论】本研究证实SacpolⅣ具有很强的跨越损伤合成能力,能够跨越多种氢键配对能力减弱的损伤碱基,为其在细胞内的跨越损伤合成功能提供了生化证据。     

人淋巴细胞经冻存复苏后DNA,RNA和蛋白质的合成代谢

本实验采用同位素掺入和光镜放射自显影技术,研究新鲜未经冻存、冻存1年和冷冻杀伤的人外周血淋巴细胞,对3~H-TdR、3~H-UR和3~H-亮氨酸的掺入量进行测量。并观察其掺入部位,以反映淋巴细胞经过冻存不同时间后,它们的DNA、RNA和蛋白质的合成强度和合成部位。实验结果表明,冻存1年的淋巴细胞,复苏后的回收率和存活率均在90％以上。PHA刺激后的3~H-TdR、3~H-UR和3~H-亮氨酸的掺入量,虽比冻存前降低,但仍相当活跃,保持较高水平。3~H-TdR集中在细胞核,3~H-UR和3~H-亮氨酸除分布在细胞质外,在核内也有一定数量。表明在合适的冻存条件下,长期深低温冻存的淋巴细胞,能保持较高的DNA、RNA和蛋白质合成代谢水平。     

NAMPT与年龄相关性疾病的研究进展

烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase,NAMPT)是哺乳动物NAD~+生物合成中的限速酶,因此是细胞内NAD~+水平的控制器。NAMPT介导的NAD~+的生物合成在能量代谢、DNA修复、染色质重塑、细胞衰老和免疫细胞功能调节等方面发挥重要的作用。然而NAMPT的循环水平随着年龄的增长而显著下降,导致年龄相关性疾病包括代谢性疾病、神经退行性疾病、衰老和癌症的发生。最近研究发现,通过脂肪组织过表达eNAMPT来提高NAD~+水平可延长小鼠的健康寿命。因此推测NAMPT-NAD~+是一种有前景的抗衰老干预途径。该文系统概述了NAMPT,总结其与年龄相关性疾病的研究进展,NAMPT作为一种具有临床意义的分子,在年龄相关性疾病的诊断、预后和治疗中具有广泛的应用前景。     

核苷酸的生物学功能

核苷酸除了构成遗传信息的基础DNA和RNA以外,在活细胞内,大量的核苷酸在物质和能量的代谢中还显示出多种多样的生物学功能. (一)重要辅酶的构件 1.是辅酶Ⅰ(NAD~+)和辅酶Ⅱ(NADP~+)的组分:腺苷酸是NAD~+和NADP~+的组成成分.NAD~+和NADP~+是细胞内很多脱氢酶的辅酶,是重要的载氢体,能传递质子和电子:2H=2H~+(质子)+2e(电子)     

EDU 免疫荧光法检测整合素连接激酶（ILK）高表达 对新生大鼠心肌细胞DNA 合成的影响

目的:通过EDU免疫荧光法观察整合素连接激酶(Integrin-Linked Kinase,ILK)在新生大鼠心肌细胞内高表达后对心肌细胞DNA合成的影响。方法:取新生(1-3天内)大鼠原代心肌细胞,培养72小时后随机分为正常对照组、ILK转染组。对照组转染重组腺病毒载体(adeno-GFP),ILK组转染重组腺病毒载体+ILK基因(adeno-ILK)。转然成功后48小时将两组心肌细胞分别通过5-乙基-2’-脱氧尿嘧啶核苷(EDU)免疫荧光法测定心肌细胞DNA合成。结果:ILK转染组心肌细胞内DNA合成较对照组明显增加(P<0.05)。结论:ILK高表达具有促进新生大鼠心肌细胞的DNA合成的能力。     

5-氮胞苷对贵州小型猪淋巴细胞DNA损伤及修复的影响

目的　研究贵州小型猪淋巴细胞对化学物或药物引起的DNA损伤及修复影响的反应。方法　用单细胞凝胶电泳技术检测比较 5 氮胞苷对PHA刺激和未刺激淋巴细胞的DNA损伤及其修复过程。结果　 5 氮胞苷引起未刺激淋巴细胞明显的DNA泳动 (彗星尾 ) ,经修复孵育 2h后 ,DNA泳动与孵育前比较无显著差异 ,而 5 氮胞苷引起的刺激细胞DNA泳动经 2h修复孵育后与孵育前比较显著减少。结论　 5 氮胞苷引起贵州小型猪未刺激淋巴细胞DNA损伤经 2h孵育未能修复 ,而刺激细胞的DNA损伤明显修复。     

DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10(-3) M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (3H-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated G0 lymphocytes, UV-irradiated or unirradiated, an increased incorporation of 3H-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of 3H-TdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10(-3)-10(-2) M NTA.     

Unscheduled DNA synthesis induced by N-acetoxy-2-acetylaminofluorene is not sensitive to regulation by ADP-ribosyl transferase

We have directly compared in resting human mononuclear leukocytes the DNA repair effects caused by ADP-ribosyl transferase (ADPRT) activity following DNA damage induction by gamma radiation, UV radiation, ethylene oxide (EO) and N-acetoxy-2-acetylaminofluorene (NA-AAF). The presence of inhibitors of ADPRT during the quantitation of unscheduled DNA synthesis (UDS) resulted in about a 2-fold increase of UDS when induced by gamma radiation, UV radiation or EO. The stimulation of UDS by EO, UV- or gamma-radiation in the presence of an ADPRT inhibitor was equally strong whether 1 mM or 10 mM hydroxyurea was used to suppress scheduled DNA synthesis. The level of NA-AAF induced UDS was not affected by inhibitors of ADPRT. In addition, direct estimation of ADPRT activity revealed that at doses giving maximal UDS, NA-AAF damage did not induce a measurable enzymatic activity whereas gamma-radiation, UV radiation and EO all showed a significant dose response increase. We have interpreted our data to mean that NA-AAF induced UDS estimates DNA repair relating mainly to DNA lesions that are recognized with difficulty, and hence, the rate of endonuclease-induced DNA strand break accumulation is not sufficient to allow a stimulation of ADPRT and affect the quantitation of UDS.     

Ultraviolet light sensitivity,unscheduled DNA synthesis and DNA repair in C7-10 Aedes albopictus mosquito cells

We have examined the relative sensitivity of Aedes albopictus C7-10 mosquito cells to irradiation with ultraviolet light from a germicidal lamp. On the basis of plating efficiency, C7-10 cells were approximately two times more resistant to UV light than human 293 leukemia cells. Recovery after UV irradiation was accompanied by an increase in unscheduled DNA synthesis (UDS), which was measured by incorporation of ³H-thymidine into acid-precipitable DNA in the presence of hydroxyurea. Under standardized conditions, UDS was maximal after a 10 min exposure (120 J/m²), and declined after longer exposures. In addition, UV treatment is associated with a small but reproducible increase in repair of plasmid DNA in transiently transfected cells. We anticipate that analysis of DNA repair activities in mosquito cells will identify molecular targets that might control longevity in transgenic mosquitoes.     

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.     

Radiation protection of stimulated human lymphocytes by nicotinamide

Nicotinamide (NA) when added to human lymphocytes in vitro together with a mitogen, protected against the inhibition by gamma and UV radiation of stimulated cell growth. When stimulated by phytohemagglutinin (PHA), concanavalin A (Con A) or pokeweed mitogen (PWM) maximum protection has been observed with approximately 1 mM NA (dose reduction factor of 2-3). To obtain protection the cells had to be stimulated immediately after irradiation in the presence of NA. It is suggested that the intracellular level of NAD+ may be rate limiting for excision repair in human lymphocytes irradiated in the G0 phase. This level is presumably increased by exogenously supplied NA, leading to enhanced repair of DNA damage and increased survival.     

Effects of in vivo exposure to antineoplastic drugs on DNA repair and replication in human lymphocytes

In peripheral blood lymphocytes of 12 nurses and 3 patients exposed to antineoplastic drugs we determined the ability to repair DNA after UV irradiation and DNA replicative synthesis after stimulation by PHA. In nurses the levels of unscheduled DNA synthesis and DNA replication were not different than in a control group, whereas in patients significant changes were observed during and after chemotherapy in the level of both types of DNA synthesis.     

Poly(ADP-ribose) Polymerase inhibitors preserve nicotinamide adenine dinucleotide and adenosine 5'-triphosphate pools in DNA-damaged cells: mechanism of stimulation of unscheduled DNA synthesis

Inhibitors of poly(ADP-ribose) polymerase stimulated the level of DNA, RNA, and protein synthesis in DNA-damaged L1210 cells but had negligible effects in undamaged L1210 cells. The poly(ADP-ribose) polymerase inhibitors stimulated DNA repair synthesis after cells were exposed to high concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (68 and 136 microM) but not after exposure to low concentrations (13.6 and 34 microM). When the L1210 cells were exposed to 136 microM N-methyl-N'-nitro-N-nitrosoguanidine, the activation of poly(ADP-ribose) polymerase resulted in the rapid depletion of oxidized nicotinamide adenine dinucleotide (NAD+) levels and subsequent depletion of adenosine 5'-triphosphate (ATP) pools. After low doses of N-methyl-N'-nitro-N-nitrosoguanidine (13.6 microM), there were only small decreases in NAD+ and ATP. Poly(ADP-ribose) polymerase inhibitors prevented the rapid fall in NAD+ and ATP pools. This preservation of the ATP pool has a permissive effect on energy-dependent functions and accounts for the apparent stimulation of DNA, RNA, and protein synthesis. Thus, the mechanism by which poly(ADP-ribose) polymerase inhibitors stimulate DNA, RNA, and protein synthesis in DNA-damaged cells appears to be mediated by their ability to prevent the drastic depletion of NAD+ pools that occurs in heavily damaged cells, thereby preserving the cells' ability to generate ATP and maintain energy-dependent processes.     

DNA repair in human cells: in Cockayne syndrome cells rejoining of DNA strands is impaired

Fibroblasts from patients with Cockayne Syndrome (CS) are hypersensitive to UV light. DNA repair was analyzed in these cells by sedimentation behaviour of DNA nucleoids in sucrose gradients and compared to normal control cells. The initiation of repair, the incision of the DNA strand next to the UV lesion appeared to be normal. The rejoining of DNA stretches, however, is retarded in CS cells. DNA repair synthesis of UV damages was measured by autoradiography of [14C]thymidine incorporation into resting cells. Up to 4 h the DNA repair synthesis was comparable with normal cells. From 4 to 7 h the incorporation of radioactive precursors declined in CS cells. Besides a defective DNA polymerase this could be due to accelerated excorporation of radioactive nucleotides as a consequence of delayed ligation. In ligation the enzyme itself could be affected as well as its activation by ADP-ribosylation. Nicotine adenine dinucleotide (NAD+) is needed for the ADP ribosylation process. The cellular NAD+ content, however, was found to be the same in normal and in CS fibroblasts. Increase of the extracellular NAD+ supply accelerated the rejoining of UV damaged DNA in CS cells.     

Xeroderma pigmentosum (complementation group D) mutation is present in patients affected by trichothiodystrophy with photosensitivity

Summary We studied the response to UV irradiation in cells from four patients, from three apparently unrelated families, affected by trichothiodystrophy (TTD). They showed all the symptoms of this rare autosomal recessive disorder (brittle hair with reduced sulfur content, mental and physical retardation, ichthyosis, peculiar face) together with photosensitivity. We found a decreased rate of duplicative DNA synthesis in stimulated lymphocytes, reduced survival in fibroblasts, and very low levels of unscheduled DNA synthesis (UDS) in Go lymphocytes and fibroblasts after UV irradiation. Complementation studies showed that normal values of UDS are restored in heterokaryons obtained by fusion of TTD cells with normal and xeroderma pigmentosum (XP)-complementation group A-cells. In contrast the defect is not complemented by fusion with XP-complementation group D-fibroblasts.     

Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase delta as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, we describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, alpha or delta. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase alpha several hundred times more strongly than it inhibits DNA polymerase delta. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase delta. It appears that repair synthesis at late times after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase delta.