人细胞端粒DNA复制与长度维持

端粒位于染色体末端,由短的串联重复DNA片段及其结合蛋白组成。端粒在维持基因组稳定性及染色体结构完整性方面发挥着重要作用。端粒DNA由富含G/C的序列构成,包括双链区及G含量高的3'悬垂单链区(G-overhang,G-tail)。端粒DNA能够形成G四联体(G-quadruplex)和T环(T-loop)等高级结构。许多与DNA损伤修复相关的蛋白质参与端粒DNA的复制与端粒结构的维持,并相对于基因组的其他区域,端粒的DNA复制较为特别,从广义上讲,端粒DNA的复制可以包括双链复制(telomere replication),端粒酶复制延伸(telomerase extension)和C链补齐(C-rich fill-in)。端粒双链复制引起的端粒长度缩短是导致人体细胞衰老的重要原因,而端粒酶复制延伸及C链补齐是干细胞及肿瘤细胞维持其端粒长度及持续分裂能力的主要途径。端粒复制及其结构功能研究是生物医学领域的一个重要热点,阐释端粒复制的机理将为疾病预防及治疗等提供新的思路。     

体外三链辫结DNA的原子力显微镜证据

采用原子力显微镜(AFM)观察了λ-DNA/HindⅢ体外加热变性后,快速冷却所形成的一种对DNaseⅠ酶有抗性的新结构DNA.其表观宽度和高度明显不同于同条件下测定的双螺旋DNA.将该DNA样品再次加热变性后,可观察到局部形成的三链辫结结构.因此,认为这种新结构DNA可能是一种有序的三链辫结DNA.     

五个链霉菌线型质粒端粒的克隆和分析

本室从西藏采集的土壤样品中分离到了一批链霉菌,利用脉冲电泳确定了其中5株链霉菌含有较小的线型质粒。【目的】克隆、测序和分析5个线型质粒的端粒。【方法】采用改良的"在凝胶中进行DNA碱处理和限制性内切酶酶切"的方法来克隆线型质粒的端粒DNA。【结果】克隆和测序了5个线型质粒的端粒DNA。通过与链霉菌典型端粒进行比较,发现这5个新的线型质粒的端粒序列同样含有多个回文序列。但是有的端粒保守的回文序列I不一定能够"折返"与内部序列配对形成"超级发卡"结构,回文序列的"突出环"不一定都为3nt。【结论】采用改良的方法克隆和鉴定了5个线型质粒新的端粒序列,这些新端粒的特征暗示:回文序列I的"折返"和3nt的回文序列的"突出环"不是端粒复制必需的。     

染色体端粒研究进展

染色体端粒(telomere)是真核生物线性染色体两端的特殊DNA--蛋白质复合体结构,由随机重复序列组成的DNA序列和与之相结合的蛋白质分子构成。端粒DNA无论在DNA顺序、功能及其特殊的复制方式都与其它DNA顺序显著不同。本文将近年来对端粒的DNA结构、与端粒DNA相结合的蛋白质分子和在端粒复制中起重要作用的反转录酶--端粒酶(telomerase)的研究进展以及端粒对于真核生物的重要作用作一综述。     

激光对DNA作用机理的AFM研究

激光作用质粒DNA和小牛胸腺DNA产生损伤效应,导致DNA结构变化,利用一种改进的试样制备过程和纳米显微镜－－原子力显微镜（AFM）能够获得可重现的激光作用质粒DNA和小牛胸腺DNA的AFM图像,显示它们的特殊的表达结构,讨论了激光辐照导致DNA链断裂的作用机理。     

老幼龄扬子鳄端粒相对长度的差异性分析

端粒（Telomere）是线性真核细胞染色体末端的一种结构,由高度重复的DNA序列和结合蛋白所构成。在脊椎动物中,端粒通常为富含TG简单重复序列（TFAGGG）,其生物学功能是完成染色体末端复制,使DNA免受不恰当的修复以及防止端一端融合和核酸外切酶的降解（Dreesen et al．,2007）。     

DNA单分子近场光学成像与荧光探测

介绍了扫描近场光学(SNOM-Scanning Near-Field Optical Microscope)／原子力显微镜（AFM－Atomic Force Microscope)系统（SNO／AM）的工作原理。在AFM模式和SNOM模式下对DNA分子进行成像和荧光探测,得到了清晰的DNA单分子的形貌像和荧光像。由形貌圆像得到的DNA分子尺寸横向为20nm,高度为2nm,其中包含了探针形貌的影响。实验中采Tapping模式的AFM成像,样品经多次搜索扫描无明显损坏。AFM模式的分辨率优于1nm。SNOM模式下DNA分子形貌像和荧光像清晰,由近场荧光分布可以确定分子取向和浓度。用YOYO－1染料对λDNA分子进行染色和荧光探测。通过对DNA分子多个截面进行测量,分析染料 与DNA结合状态。     

用EndoV和碱电泳法检测哺乳动物细胞嘧啶二聚体的切除修复

紫外线(UV)辐射是自然环境中重要的DNA致伤因子。UV辐射在DNA造成的最主要一类损伤产物是环丁烷嘧啶二聚体(pyrim-idine dimer,PD),它是由DNA中一条多核着酸链上两相邻嘧啶碱基各自的C5和C6共价连接形成的环丁烷结构。哺乳动物细胞主要通过切除修复途径移除PD,恢复DNA的正常结构。一种从噬菌体T4感染的E.coli中提取的T4核酸内切酶V(EndoV)能特异识别PD,并在该损伤位点切断磷酸二醋键,造成单链断裂。本文即以EndoV为探针,以其敏感位点(endonuclease-sensitive-site,ESS)     

大鼠脑cDNA文库的构建

采用简单高效的cDNA合成技术制备Wistar大鼠脑cDNA基因文库,以纯化的poly( A)⁺－RNA为模板,含Not I切点的oligo－（dT）15为引物,在反转录酶的作用下,合成第一股单链cDNA;用E.coli RNase H除去模板RNA,并以E.coli DNA聚合酶Ｉ,E.coli DNA连接酶和T4 DNA聚合酶催化合成cDNA第二条链,即成为双链cDNA;此双股cDNA除0．5μg用于插入pSPORT I载体,转入E.coli DH5a,建成cDNA文库外,其余保存在－20℃,以此cDNA为模板,应用PCR方法,先后克隆了谷氨酸脱羧酶（GAD,1800bp）、神经元特异性烯醇化酶（NSE,1340bp）,甲状腺激素受体（T3－receptor,1230bp）、胆囊收缩素（CCK,345bp）的全编码基因．     

游动双孢菌线型质粒pPR2的全序列测定及分析

[目的]获得游动双孢菌线型质粒pPR2的全序列,并揭示新型的端粒复制蛋白和可能的中间复制位点.[方法]用分段克隆的方法和序列拼接获得pPR2的全序列,利用软件分析端粒DNA的二级结构和可能的端粒复制蛋白,利用链霉菌原生质体转化的方法检测可能的中间复制的位点.[结果]pPR2全长为15520 bp,(G C)含量为68.1%.其端粒末端反向重复序列的长度为329 bp,不能像多数链霉菌的线型质粒那样能形成保守的"折返"的二级结构.pPR2虽然没有参与链霉菌端粒复制的保守的tap/tpg基因,但是pPR2.3c基因编码了一个双结构域蛋白,分别同链霉菌的端粒复制相关蛋白Tap和嗜血杆菌的解旋酶具有相似性.pPR2缺少典型的链霉菌重复序列-复制基因(iteron-rep)区段,将几乎覆盖全长pPR2的两段DNA进行克隆后,不能转化变铅青链霉菌.此外,pPR2基因还编码可能参与线型DNA复制的调控的单链结合蛋白(SSB)和与放线菌质粒接合转移相关的主要蛋白(Tva).[结论]pPR2是链霉菌之外的放线菌中最小的线型质粒,其序列在游动双孢菌属的线型质粒中是首次报道.pPR2可能具有新型的端粒复制的机制,其中pPR2.2c和pPR2.3c编码可能的端粒复制蛋白.     

Mutagenic SOS repair of site-specific psoralen damage in plasmid pBR322

The mutagenic repair of psoralen damage was examined by transforming Escherichia coli with psoralen-treated pBR322. Plasmid DNA randomly reacted with psoralen was repaired only when the E. coli was uvrA⁺ and recA⁺, and only when the cells were pre-irradiated with far-ultraviolet light. The recA dependence and requirement for pre-irradiation are characteristics of SOS repair.Psoralens were placed specifically near the BamHI site, in the tetracycline-resistance gene of pBR322, using a sulfhydryl-containing psoralen derivative. Repair of this damage also required pre-irradiation of the host cells. This repair was accompanied by a 4% frequency of mutagenesis to a tetraeycline-sensitive phenotype. Sequence analysis of these mutant plasmids revealed that 75% had mutations within the targeted region, while 25% had no sequence changes within 100 bases of the BamHI site. In up to five independent isolates only one kind of mutation was observed at each site, suggesting that mutagenic SOS repair is influenced by DNA structure at the site of the psoralen. Most mutations were transitions, primarily G-C to A-T changes. Some transitions occurred at sites where psoralen crosslinks could not have formed, and these may have arisen from the repair of psoralen monoadducts.     

Stability of DNA triplexes on shuttle vector plasmids in the replication pool in mammalian cells

Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.     

Processing of targeted psoralen cross-links in Xenopus oocytes.

Psoralen cross-links have been shown to be both mutagenic and recombinagenic in bacterial, yeast, and mammalian cells. Double-strand breaks (DSBs) have been implicated as intermediates in the removal of psoralen cross-links. Recent work has suggested that site-specific mutagenesis and recombination might be achieved through the use of targeted psoralen adducts. The fate of plasmids containing psoralen adducts was evaluated in Xenopus oocytes, an experimental system that has well-characterized recombination capabilities and advantages in the analysis of intermediates in DNA metabolism. Psoralen adducts were delivered to a specific site by a triplex-forming oligonucleotide. These lesions are clearly recognized and processed in oocytes, since mutagenesis was observed at the target site. The spectrum of induced mutations was compared with that found in similar studies in mammalian cells. Plasmids carrying multiple random adducts were preferentially degraded, perhaps due to the introduction of DSBs. However, when DNAs carrying site-specific adducts were examined, no plasmid loss was observed and removal of cross-links was found to be very slow. Sensitive assays for DSB-dependent homologous recombination were performed with substrates with one or two cross-link sites. No adduct-stimulated recombination was observed with a single lesion, and only very low levels were observed with paired lesions, even when a large proportion of the cross-links was removed by the oocytes. We conclude that DSBs or other recombinagenic structures are not efficiently formed at psoralen adducts in Xenopus oocytes. While psoralen is not a promising reagent for stimulating site-specific recombination, it is effective in inducing targeted mutations.     

Defective replication of psoralen adducts detected at the gene-specific level in xeroderma pigmentosum variant cells.

Replication of damaged DNA is suspected to play an important role in cell cycle, genetic stability, and survival pathways. Using psoralen photoaddition as prototype DNA damage and the renaturing agarose gel electrophoresis technique to measure DNA cross-linking in individual genes, Vos and Hanawalt previously observed efficient bypass replication of psoralen monoadducts in human genes (J.-M. H. Vos and P. C. Hanawalt, Cell 50:789-799, 1987). To understand the mechanism of bypass replication in human cells, mutants affected in such a process would be useful. We now report that cells from individuals suffering from the hereditary recessive syndrome xeroderma pigmentosum variant (XPV) are hypersensitive to killing induced by photoactivated psoralen. In addition, analysis of psoralen-mediated DNA cross-linking in the rRNA genes indicated that although repair of psoralen adducts was similar to that of normal individuals, XPV cells were markedly deficient in the ability to bypass psoralen adducts during replication; in comparison with normal cells, approximately half as many monoadducts were bypassed during replication in XPV cells. Furthermore, in contrast to normal cells, replication of interstrand cross-links was not detected in XPV. This is the first demonstration of a deficiency in bypass replication detected at the gene-specific level in vivo. A model involving a strand-specific defect in recombinational bypass in XPV is proposed.     

High resolution psoralen mapping reveals an altered DNA helical structure in the SV40 regulatory region.

Preferential psoralen photobinding sites have been mapped in vitro on restriction fragments spanning the SV40 origin region and surrounding sequences by a new fine structure analysis technique. Purified DNA fragments were photoreacted with 3H-5-methylisopsoralen (3H-5-MIP), a psoralen derivative which forms only monoadducts. Fragments were then end-labeled and digested with lambda exonuclease, a 5' processive enzyme which we have determined pauses at 5-MIP monoadducts. When photobinding sites were mapped on denaturing sequencing gels, it was observed that 5-MIP binds preferentially to 5'-TA sites, and to a lesser degree to 5'-AT sites. Utilizing this approach, we have identified a psoralen hypersensitive region in which the binding sites were much stronger than those in the surrounding sequences. This region extends from 150 base pairs (bp) to the late side of the enhancers to the early enhancer/promoter boundary. We suggest that this region contains a sequence directed structural alteration of the DNA helix which can be detected by the psoralen mapping approach described.     

Psoralen-DNA crosslink repair in human lymphocytes: Comparison of alkaline elution with electron microscopy

Much interest has surrounded the question of the removal of psoralen interstrand crosslinks in DNA of eukaryotic organisms. A commonly employed method for the study of psoralen repair is alkaline elution. In this study we have used alkaline elution to assess psoralen crosslink repair in human lymphocytes. The lymphocytes were treated with 8-methoxypsoralen or 4,5′,8-trimethylpsoralen and allowed to repair for different periods of time. Analysis by alkaline elution showed elution patterns compatible with crosslink removal. When the crosslink removal under comparable conditions was studied by the use of electron microscopy under totally denaturing conditions, no repair of the crosslinks could be detected.     

Nucleosome assembly in mammalian cell extracts before and after DNA replication.

Protein-free DNA in a cytosolic extract supplemented with SV40 large T-antigen (T-Ag), is assembled into chromatin structure when nuclear extract is added. This assembly was monitored by topoisomer formation, micrococcal nuclease digestion and psoralen crosslinking of the DNA. Plasmids containing SV40 sequences (ori- and ori+) were assembled into chromatin with similar efficiencies whether T-Ag was present or not. Approximately 50-80% of the number of nucleosomes in vivo could be assembled in vitro; however, the kinetics of assembly differed on replicated and unreplicated molecules. In replicative intermediates, nucleosomes were observed on both the pre-replicated and post-replicated portions. We conclude that the extent of nucleosome assembly in mammalian cell extracts is not dependent upon DNA replication, in contrast to previous suggestions. However, the highly sensitive psoralen assay revealed that DNA replication appears to facilitate precise folding of DNA in the nucleosome.     

Human XPC-hHR23B interacts with XPA-RPA in the recognition of triplex-directed psoralen DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair (NER), XPA-RPA, recognizes DNA ICLs. We now report the use of triplex technology to direct a site-specific psoralen ICL to a target DNA substrate to determine whether the human global genome NER damage recognition complex, XPC-hHR23B, recognizes this lesion. Our results demonstrate that XPC-hHR23B recognizes psoralen ICLs, which have a structure fundamentally different from other lesions that XPC-hHR23B is known to bind, with high affinity and specificity. XPC-hHR23B and XPA-RPA protein complexes were also observed to bind psoralen ICLs simultaneously, demonstrating not only that psoralen ICLs are recognized by XPC-hHR23B alone, but also that XPA-RPA may interact cooperatively with XPC-hHR23B on damaged DNA, forming a multimeric complex. Since XPC-hHR23B and XPA-RPA participate in the recognition and verification of DNA damage, these results support the hypothesis that interplay between components of the global genome repair sub-pathway of NER is critical for the recognition of psoralen DNA ICLs in the mammalian genome.