增殖细胞核抗原（PCNA）的分子结构及其生物学功能研究进展

增殖细胞核抗原（PCNA）是真核生物复制复合体的核心成分，具有特殊的环状三级结构. 作为真核细胞DNA聚合酶δ的推动因子，与不同复制相关蛋白结合，协调DNA复制过程. 同时PCNA还作为功能转换因子，通过不同调控方式与多种细胞因子作用，参与了DNA损伤修复、细胞周期调控及凋亡等许多重要的细胞事件. 另外作为细胞增殖的指标，PCNA与肿瘤等细胞增殖性疾病的发生和发展存在相关性，因此在临床上对PCNA的深入研究有重要意义. 文中就PCNA的“功能性”结构及其在不同细胞事件中的功能转换（Function Switch）进行简要综述.     

真核生物DNA聚合酶δ的研究现状

DNA聚合酶δ(DNA polymerase δ)是真核生物DNA复制的主要复制酶,同时还参与DNA修复,对保持真核生物基因组的结构完整性和遗传稳定性具有重要作用.对DNA聚合酶δ蛋白功能活性及其基因表达机制的研究因受技术上的限制而未能深入研究.由于其重要的生物学功能,目前引起人们的更多关注和重视.文中就该酶的生物学功能、亚基组成、核心酶的分子表达调控以及与其他蛋白相互作用等方面对国内外DNA聚合酶δ的研究进行简要综述.     

NuA4复合体及其在细胞内的功能

NuA4复合体是进化中非常保守的多亚基复合体。在真核生物中，NuA4复合体及其同源复合体广泛参与了基因转录激活、DNA损伤修复、细胞周期、糖代谢等重要的细胞生理过程。由于NuA4复合体具有乙酰化组蛋白H4／H2A和激活转录的能力而被人们广泛研究。对NuA4及其同源复合体组成和功能方面的研究进行简要介绍。     

高等植物中的DNA解旋酶

综述了在离体实验条件下高等植物DNA解旋酶在以下多方面具有重要的生物学功能：包括DNA重组、DNA复制、翻译启始、rDNA转录及在prerRNA加工过程早期阶段,双链断裂修复、端粒长度维持、核苷酸切除修复、花发育中的细胞分裂/增殖、基因组甲基化方式保持、植物细胞周期和细胞基本生命活动的维持。最近玉米基因组中发现的解旋子（helitron)插入说明高等植物DNA解旋酶可能在植物生长与发育中有重要调控作用。因而有着重要的生物技术应用价值。     

DNA拓扑异构酶Ⅱβ结合蛋白1（TopBP1）磷酸化功能位点推测及功能分析

为阐明DNA拓扑异构酶Ⅱβ结合蛋白1(TopBP1)参与DNA损伤修复应答的分子机制，本研究通过生物信息学分析，发现多个潜在的TopBP1磷酸化位点T860，S887，T1104及T1167，利用分子生物学手段从人cDNA文库中扩增并获得TopBP1克隆质粒，将上述磷酸化位点突变为丙氨酸，观察突变质粒转染细胞对DNA损伤修复的应答反应. 结果显示，在粒子射线照射或化疗药物处理细胞后，第1104丙氨酸突变（T1104A） 的TopBP1蛋白导致pRPA32-S33的磷酸化水平大幅降低，同时细胞周期检验点失活，严重阻滞了DNA应激反应，证实T1104是TopBP1参与DNA损伤修复的关键活性位点.     

高等植物中的DNA解旋酶

综述了在离体实验条件下高等植物DNA解旋酶在以下多方面具有重要的生物学功能：包括DNA重组、DNA复制、翻译启始、rDNA转录及在pre-rRNA加工过程早期阶段，双链断裂修复、端粒长度维持、核苷酸切除修复、花发育中的细胞分裂／增殖、基因组甲基化方式保持、植物细胞周期和细胞基本生命活动的维持。最近玉米基因组中发现的解旋子(helitron)插入说明高等植物DNA解旋酶可能在植物生长与发育中有重要调控作用。因而有着重要的生物技术应用价值。     

基于原子力显微镜研究p53蛋白与PBR322 DNA的相互作用

肿瘤(癌症)是由于细胞在复制过程中,DNA损伤不能修复导致细胞凋亡或者细胞无限增殖而形成的。DNA在细胞中转录和翻译都会涉及蛋白与DNA的结合,肿瘤抑制蛋白也是参与这一过程的关键蛋白之一。然而,众多研究发现,肿瘤抑制蛋白p53具有识别和修复损伤DNA的效果,对于细胞的凋亡、基因的保护和避免癌症发生有着重要的意义。有研究表明,金属镁离子和锌离子可以增强p53蛋白的结构稳定性和p53-DNA的亲和力。因此,我们基于原子力显微镜(AFM),直观地呈现出p53蛋白与PBR322环状DNA相互作用的图像,同时发现p53蛋白可以使环状DNA自身形成聚集或者相交。但是,对于长度相当的5 000bp的线状DNA几乎没有这样的效果,而对于20 000bp DNA不会出现这样的现象。然而,在高浓度镁离子环境下,环状DNA会扭转成为麻花状,即形成超螺旋结构。这一现象,可为p53蛋白功能和作用机理研究提供指导,也为癌症治疗、癌症药物开发以及癌症检测方法提供启发。     

香兰素衍生物VND3207对α粒子辐射诱发人成纤维细胞基因组DNA损伤的防护研究

 高能α粒子辐射所致基因组DNA损伤形式为复杂的簇集损伤,修复难度大,生物效应严重,目前尚缺乏有效防护药物。前期发现香兰素衍生物VND3207具有抗氧化损伤、促进DNA双链断裂损伤修复的作用,能有效防护低传能线密度（LET） γ射线诱发细胞凋亡和基因组损伤。本研究旨在探讨VND3207对高能α粒子辐射诱发人成纤维细胞基因组DNA损伤的防护效应。细胞克隆形成实验表明,VND3207无论是照射前加药还是照射后加药作用均能增强人成纤维HFS细胞对α粒子的辐射损伤抗性。通过中性单细胞凝胶电泳（彗星电泳）、γ-H2AX免疫荧光簇集点、γ-H2AX蛋白表达水平、细胞微核等多个基因组DNA损伤指标分析,表明了VND3207对α粒子辐射诱发细胞基因组DNA损伤的有效防护作用,其在5 ~ 10μmol/L浓度下,就可产生显著辐射防护效果,既能减轻α粒子辐射诱发细胞DNA的原初损伤水平,又能提高细胞的DNA修复能力。因此,VND3207具有维护高能粒子辐照细胞的基因组稳定性的作用,有开发成为抗高LET辐射损伤新药的价值。     

DNA损伤和修复的生物标记物的探讨

某些物理化学因子，如紫外线，电离辐射和化学诱变剂等，都有引起生物突变和致死的作用。这些物理化学因子均能作用于DNA，造成其结构和功能的破坏。熙而在一定条件下，生物泪L体能使其DNA的损伤得到修复。本文主要介绍几种修复系统和生物标记物。     

番茄DNA复制因子RFC1在减数分裂重组中的功能研究

减数分裂是真核生物有性生殖所必需,前期研究表明拟南芥DNA复制因子RFC1在减数分裂重组中具有重要作用,但其同源基因在其他物种中的减数分裂或其他功能还未有报道.为了检验番茄RFC1在减数分裂中的功能,我们建立了减数分裂特异RFC1~(RNAi)转基因番茄,发现其花粉活性显著降低,而且减数分裂异常.利用DAPI染色观察染色体形态,发现RFC1~(RNAi)减数分裂细胞在终变期和中期Ⅰ形成多价体,表明重组异常.DNA双链断裂指示蛋白γH2AX在RFC1~(RNAi)粗线期染色体上分布的保留滞后,进一步支持RFC1~(RNAi)中的重组异常.拟南芥和番茄分别属于真双子叶植物中的两个不同大支,蔷薇类和菊类,因此本文的结果也表明DNA合成基因RFC1在进化距离很远的不同植物物种中的功能相对保守.     

Chromosome length influences replication-induced topological stress

During chromosome duplication the parental DNA molecule becomes overwound, or positively supercoiled, in the region ahead of the advancing replication fork. To allow fork progression, this superhelical tension has to be removed by topoisomerases, which operate by introducing transient DNA breaks. Positive supercoiling can also be diminished if the advancing fork rotates along the DNA helix, but then sister chromatid intertwinings form in its wake. Despite these insights it remains largely unknown how replication-induced superhelical stress is dealt with on linear, eukaryotic chromosomes. Here we show that this stress increases with the length of Saccharomyces cerevisiae chromosomes. This highlights the possibility that superhelical tension is handled on a chromosome scale and not only within topologically closed chromosomal domains as the current view predicts. We found that inhibition of type I topoisomerases leads to a late replication delay of longer, but not shorter, chromosomes. This phenotype is also displayed by cells expressing mutated versions of the cohesin- and condensin-related Smc5/6 complex. The frequency of chromosomal association sites of the Smc5/6 complex increases in response to chromosome lengthening, chromosome circularization, or inactivation of topoisomerase 2, all having the potential to increase the number of sister chromatid intertwinings. Furthermore, non-functional Smc6 reduces the accumulation of intertwined sister plasmids after one round of replication in the absence of topoisomerase 2 function. Our results demonstrate that the length of a chromosome influences the need of superhelical tension release in Saccharomyces cerevisiae, and allow us to propose a model where the Smc5/6 complex facilitates fork rotation by sequestering nascent chromatid intertwinings that form behind the replication machinery.     

肿瘤研究新视点——MGMT

MGMT作为一种重要的DNA修复酶对于保护细胞免受环境中的致癌物质和（或）化疗物引起的基因毒起着关键作用，因此在细胞对环境致癌物的易感性与肿瘤对化学药物的耐药性等研究领域，MGMT正受到越来越多研究者的关注。化疗药物作用的有限性一直是困扰肿瘤临床治疗的一大难题，许多人把希望寄托在肿瘤的早期发现与预防上，MGMT作为一种重要的DNA修复酶起着保护细胞免受基因毒的重要作用，利用这一性质，通过检测组织中MGMT的活性水平可以帮助我们预测正常细胞的突变性与肿瘤细胞的耐药性，为肿瘤的早期预防和确定化疗方案提供依据，本文综述了MGMT的一般性质、作用机制和临床意义。     

Role of Tip60 tumor suppressor in DNA repair pathway

To prevent the damage caused by DNA strand breaks, eukaryotic cells have evolved a series of highly conserved DNA repair mechanisms. The ubiquitously expressed acetyltransferase, Tip60, plays a central role in ATM (ataxia-telangiectasia mutated) activation which is involved in DNA repair. Recent work uncovered a new mechanism of ATM activation mediated by Tip60 and demonstrated that histone methylation, specifically, trimethylation of histone H3, is a key factor in the process. Here, we review the current understanding of how Tip60 is activated and how it activates ATM in response to DNA damage.     

Molecular structure and biological function of proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) is the core component of replication complex in eukaryote. As a processive factor of DNA polymerase delta, PCNA coordinates the replication process by interacting with various replication proteins. PCNA appears to play an essential role in many cell events, such as DNA damage repair, cell cycle regulation, and apoptosis, through the coordination or organization of different partners. PCNA is an essential factor in cell proliferation, and has clinical significance in tumor research. In this article we review the functional structure of PCNA, which acts as a function switch in different cell events.     

Molecular structure and biological function of proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) is the core component of replication complex in eukaryote. As a processive factor of DNA polymerase delta, PCNA coordinates the replication process by interacting with various replication proteins. PCNA appears to play an essential role in many cell events, such as DNA damage repair, cell cycle regulation, and apoptosis, through the coordination or organization of different partners. PCNA is an essential factor in cell proliferation, and has clinical significance in tumor research. In this article we review the functional structure of PCNA, which acts as a function switch in different cell events.     

Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA

How DNA repair proteins distinguish between the rare sites of damage and the vast expanse of normal DNA is poorly understood. Recognizing the mutagenic lesion 8-oxoguanine (oxoG) represents an especially formidable challenge, because this oxidized nucleobase differs by only two atoms from its normal counterpart, guanine (G). Here we report the use of a covalent trapping strategy to capture a human oxoG repair protein, 8-oxoguanine DNA glycosylase I (hOGG1), in the act of interrogating normal DNA. The X-ray structure of the trapped complex features a target G nucleobase extruded from the DNA helix but denied insertion into the lesion recognition pocket of the enzyme. Free energy difference calculations show that both attractive and repulsive interactions have an important role in the preferential binding of oxoG compared with G to the active site. The structure reveals a remarkably effective gate-keeping strategy for lesion discrimination and suggests a mechanism for oxoG insertion into the hOGG1 active site.     

洋葱染色体螺旋与放射环结构的电镜观察

染色体经DNA特异染色后,观察到其最高层次结构是螺旋的,而次级结构存在着放射环结构,即由直径为25～30nm的DNA纤丝放射状折叠排列成约250nm左右的染色单体丝;染色单体丝进一步螺旋形成中期染色体结构。这些观察结果和染色体的螺旋与放射环共存模型非常相似。这个模型应更接近于洋葱染色体真实结构。     

Frequent chromosomal translocations induced by DNA double-strand breaks

The faithful repair of DNA damage such as chromosomal double-strand breaks (DSBs) is crucial for genomic integrity. Aberrant repair of these lesions can result in chromosomal rearrangements, including translocations, which are associated with numerous tumours. Models predict that some translocations arise from DSB-induced recombination in differentiating lymphoid cell types or from aberrant repair of DNA damage induced by irradiation or other agents; however, a genetic system to study the aetiology of these events has been lacking. Here we use a mouse embryonic stem cell system to examine the role of DNA damage on the formation of translocations. We find that two DSBs, each on different chromosomes, are sufficient to promote frequent reciprocal translocations. The results are in striking contrast with interchromosomal repair of a single DSB in an analogous system in which translocations are not recovered. Thus, while interchromosomal DNA repair does not result in genome instability per se, the presence of two DSBs in a single cell can alter the spectrum of repair products that are recovered.     

Dynamic analysis of DNA damage induced miRNAs in colon cancer cells

It is known that microRNAs （miRNAs） expression profile shows substantial changes in cells under DNA damage. Here, we did miRNA microarray and quantitative real-time PCR to comprehensively identify the differentially expressed miRNAs in colon cancer cell lines HCT116 p53＋/＋ and HCT116 p53-/-. Cluster analysis revealed a panel of differentially expressed miRNAs which are regulated by p53 and/or UV-C induced DNA damage. These altered miRNAs tend to be located in chromosomes 13, X and 17. Moreover, pathways enrichment analysis estimated that MAPK pathway, focal adheren pathway, p53 pathway and Wnt pathway were mediated by these miRNAs to exert their functions in DNA damage response. Additionally, we found that miR- 320a, one of the UV-C induced miRNAs, play a role in protecting cells from DNA damage. Taken together, our results show that miRNAs are dynamic regulated in p53- dependent or -independent manners in different cell contexts and different situations following DNA damage.     

RadA： A protein involved in DNA damage repair processes of Deinococcus radiodurans R1

RadA is highly conserved in bacteria and belongs to the RecA/RadA/Rad51 protein su-perfamily found in bacteria,archaea and eukarya. In Archaea,it plays a critical role in homologous re-combination process due to its RecA-like function. In Escherichia coli,it takes part in conjugational recom-bination and DNA repair but is not as important as that of archaea. Using PSI-BLAST searches,we found that Deinococcus radiodurans RadA had a higher similarity to that of bacteria than archaea and eukarya. Disruption of radA gene in D. radiodurans resulted in a modestly decreased resistance to gamma radiation and ultraviolet,but had no effect on the resistance to hydrogen peroxide. Complementa-tion of the radA disruptant by both E. coli radA and D. radiodurans radA could fully restore its resistance to gamma radiation and ultraviolet irradiation. Further domain function analyses of D. radiodurans RadA showed that the absence of the zinc finger domain resulted in a slightly more sensitive phenotype to gamma and UV radiation than that of the radA mutant,while the absence of the Lon protease domain exhib-ited a slightly increased resistance to gamma and UV radiation. These data suggest that D. radiodurans RadA does play an important role in the DNA damage repair processes and its three different domains have different functions.