DNA光修复酶修复紫外损伤的DNA

DNA光修复酶在蓝光驱动下,利用黄素腺嘌呤二核苷酸（FAD）分子的黄素酶作为催化辅助因子,来修复紫外线诱导的环丁烷嘧啶二聚体（CPD）和嘧啶（6-4）嘧啶酮的DNA损伤产物。通过无根发育树,综述了DNA光修复酶/隐花色素家族的分类;详细地阐述两种DNA光修复酶的结构、光损伤后产生的嘧啶二聚体的结构及光修复过程;最后回顾了DNA光修复酶的研究现状并展望该领域的发展前景。     

新型糖基化酶AlkC和AlkD

DNA糖基化酶是一类有着重要生物功能的蛋白质,广泛存在于原核生物和真核生物中。研究表明,DNA糖基化酶能够特异性地识别损伤碱基,再通过各种酶修复DNA。最近,科学家在筛选土壤中3-甲基腺嘌呤糖基化酶时,发现了三种基因AlkC、AlkD和AlkE,其中AlkC和AlkD是两种新基因。本文根据近年来的研究成果,对AlkC和AlkD两种碱基糖基化修复酶的结构和功能,以及AlkD的切除损伤DNA的核酸捕获机理进行了总结。     

黑腹果蝇CG18853基因编码蛋白的生物信息学分析

目的:利用生物信息学方法分析黑腹果蝇CG18853基因编码蛋白的结构和功能。方法:基于NCBI数据库中黑腹果蝇CG18853基因编码蛋白的氨基酸序列,从蛋白质的理化性质、跨膜区、信号肽、亚细胞定位、结构域、三维结构及不同物种间同源蛋白进化关系等方面进行分析。结果:果蝇CG18853蛋白的理论分子量约38.5 kDa,理论等电点为8.80。CG18853蛋白为不稳定亲水性蛋白,无跨膜区和信号肽,具有DNA光修复酶FAD结合结构域。果蝇CG18853蛋白与模板3umv.1.A有60.87%的氨基酸序列一致;CG18853蛋白与长鼻袋鼠、金鱼、拟南芥、粳稻的编码产物高度同源。结论:黑腹果蝇CG18853蛋白具有DNA光修复酶家族的典型结构,可能在细胞核中参与DNA损伤修复过程。     

O6-甲基鸟嘌呤-DNA甲基转移酶研究进展

C⁶-甲基鸟嘌呤-DNA甲基转移酶是细胞中一种重要的DNA损伤修复酶,它在抵御烷化剂所致的细胞突变和死亡中扮演重要的角色。本文综述近年来关于这个酶的研究进展,内容包括酶及其基因,酶与细胞突变,酶与肿瘤化疗的关系。     

MGMT研究进展

DNA修复酶O^6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）是机体修复烷基加合物的关键酶,它可以对抗烷化剂造成的DNA损伤,其在肿瘤发生和化疗中所起的作用对于研究肿瘤的发生及治疗均有十分重要的意义。MGMT在不同个体及组织间的表达具有极大的多样性,其表达调控机制是目前人们研究的热点。     

人造锌指蛋白的应用

C2H2锌指是真核细胞中最常见的DNA结合模体。由于C2H2锌指域靶位点特异性与结构和功能的模块性构成,使得C2H2锌指域成为构建特定的DNA结合蛋白的常用骨架。保持C2H2锌指的基本骨架不变,替换锌指特定位点的氨基酸残基,并融合表达其他功能域就可以得到具有靶向性的人造锌指蛋白(ZFP)。ZFP可以介导靶基因的转录调控,抑制或激活特定基因的表达与配体依赖的靶基因激活或抑制;对DNA进行修饰,如人造限制性内切酶,重组酶,整合酶;抗病毒感染等。因此,人造锌指蛋白应用前景广阔,研究价值显著,是未来人类基因治疗的革命性的工具。     

DNA 修复酶 MTH1 抑制剂研究进展

MTH1 是一种 DNA 氧化损伤修复酶,主要负责“清理”核苷酸池中氧化损伤的脱氧核苷三磷酸(dNTPs),以防其掺入 DNA 复 制中而造成碱基错配。研究表明,MTH1 与肿瘤细胞的生存密切相关,而正常细胞的生长与存活则不依赖于 MTH1。所以,以 MTH1 为靶 点开展抗肿瘤新药研发,已逐渐受到人们的关注。抑制 MTH1,为肿瘤治疗开辟了一条新途经。简介 MTH1 的结构和功能及其与肿瘤的关联, 着重对近年来 MTH1 抑制剂的发现过程和研究进展作一综述,探究小分子 MTH1 抑制剂与 MTH1 蛋白的作用模式,为 MTH1 抑制剂的设 计提供思路。     

WRN结构及其生物学功能研究进展

Werner综合征蛋白(Werner syndrome protein,WRN)是一种既可以和DNA结合又可以和其他蛋白质结合的具有多种酶活性的多功能DNA解螺旋酶。该酶在防止早衰与肿瘤,维持基因组完整与稳定的过程中发挥着重要的作用。文章综述了DNA解螺旋酶WRN的结构特征及其在DNA复制、DNA重组、DNA损伤修复、基因转录、维持端粒稳定、维持异染色质稳定过程中的生物学功能,并且展望了DNA解螺旋酶WRN在结构与生物学功能研究方面未来有待深入解决的问题。     

温度条件对染色体损伤修复的影响

细胞内DNA修复系统的正常工作是生物 得以生存并准确无误遗传繁衍的一个重要因 素,而修复过程的错误乃是突变发生的主要原 因。对大肠杆菌的研究证实DNA在复杂的复 制过程中自发或诱发地产生许多错误,而这些 错误最终没有形成损伤则归于各种修复过程中 酶的正常工作。一旦缺少它们,突变株就将 增加［[221。对人皮肤成纤维细胞研究发现,如 果某一修复系统缺失或被抑制,将很快导致癌 变〔1 ,9 ,10 .1370 DNA 修复系统主要包括光修复、切除修 复、重组修复、SOS修复（错误修复）、链交连修 复系统及适应性修复系统等。每一修复系统都 有若干酶参与并在细胞周期的一定时期内对一 定类型的染色体损伤进行修复。例如：人体中 常见的切除修复系统有DNA内切酶、外切酶、 多聚酶、连接酶参与,在细胞的Go, G1, G2期对 DNA的单链损伤进行识别、切割、合成、连接。 其中只要一个酶失活或受到抑制,整个修复系 统便无法正常工作。 在突变育种中,扩大突变谱提高突变率十 分重要。为此,(1)可用不同的理化因子诱发 提高DNA损伤的类型和程度,使SOS修复系 统启动,由错误修复导致突变。(2)改变修复 期环境,抑制修复酶正常活力,使DNA损伤无 法正常被修复产生突变。用某些化学物质抑制 植物修复系统中某些酶的活力,使染色体畸变 率和后代突变率增加已有报道(4,15,16,183。在哺乳 动物培养细胞中,通过升温处理可使辐射所诱 发的伤害增加[12,17,211,并抑制细胞修复,使死亡 细胞增加[11.1970 我们认为改变修复期的环境条 件,如温度、pH值、氧含量等,可能影响修复酶 的活性,导致突变率改变。本实验证实当DNA 损伤水平相同时,仅改变修复期温度,便可使细 胞突变率出现差异显著。     

结构特异性核酸酶FEN-1的功能和结构

FEN-1(flap endo/exonuclease)是一种结构特异性核酸酶,它能识别特定的DNA分叉结构,并切除含有游离5′端的单链核酸. 在DNA复制过程中,FEN-1通过其外切酶、内切酶活力去除了冈崎片段前端RNA引物的最后一个核糖核苷.在DNA修复中,FEN-1以其内切酶活力参与了损伤碱基的修复过程.FEN-1基因含有两个保守区和一个PCNA结合区.     

Uracil‐DNA glycosylases—Structural and functional perspectives on an essential family of DNA repair enzymes

Uracil‐DNA glycosylases (UDGs) are evolutionarily conserved DNA repair enzymes that initiate the base excision repair pathway and remove uracil from DNA. The UDG superfamily is classified into six families based on their substrate specificity. This review focuses on the family I enzymes since these are the most extensively studied members of the superfamily. The structural basis for substrate specificity and base recognition as well as for DNA binding, nucleotide flipping and catalytic mechanism is discussed in detail. Other topics include the mechanism of lesion search and molecular mimicry through interaction with uracil‐DNA glycosylase inhibitors. The latest studies and findings detailing structure and function in the UDG superfamily are presented.     

The structural basis of XRCC1-mediated DNA repair

Scaffold proteins play a central role in DNA repair by recruiting and organizing sets of enzymes required to perform multi-step repair processes. X-ray cross complementing group 1 protein (XRCC1) forms enzyme complexes optimized for single-strand break repair, but participates in other repair pathways as well. Available structural data for XRCC1 interactions is summarized and evaluated in terms of its proposed roles in DNA repair. Mutational approaches related to the abrogation of specific XRCC1 interactions are also discussed. Although substantial progress has been made in elucidating the structural basis for XRCC1 function, the molecular mechanisms of XRCC1 recruitment related to several proposed roles of the XRCC1 DNA repair complex remain undetermined.     

Chemical Synthesis of Oligonucleotides Containing Damaged Bases for Biological Studies

Since nucleic acids are organic molecules, even DNA, which carries genetic information, is subjected to various chemical reactions in cells. Alterations of the chemical structure of DNA, which are referred to as DNA damage or DNA lesions, induce mutations in the DNA sequences, which lead to carcinogenesis and cell death, unless they are restored by the repair systems in each organism. Formerly, DNA from bacteria and bacteriophages and DNA fragments treated with UV or γ radiation, alkylating or crosslinking agents, and other carcinogens were used as damaged DNA for biochemical studies. With these materials, however, it is difficult to understand the detailed mechanisms of mutagenesis and DNA repair. Recent progress in the chemical synthesis of oligonucleotides has enabled us to incorporate a specific lesion at a defined position within any sequence context. This method is especially important for studies on mutagenesis and translesion synthesis, which require highly pure templates, and for the structural biology of repair enzymes, which necessitates large amounts of substrate DNA as well as modified substrate analogs. In this review, the various phosphoramidite building blocks for the synthesis of lesion-containing oligodeoxyribonucleotides are described, and some examples of their applications to molecular and structural biology are presented.     

Organization of DNA damage,excision repair,and mutagenesis in chromatin: A genomic perspective

Genomic DNA is constantly assaulted by both endogenous and exogenous damaging agents. The resulting DNA damage, if left unrepaired, can interfere with DNA replication and be converted into mutations. Genomic DNA is packaged into a highly compact yet dynamic chromatin structure, in order to fit into the limited space available in the nucleus of eukaryotic cells. This hierarchical chromatin organization serves as both the target of DNA damaging agents and the context for DNA repair enzymes. Biochemical studies have suggested that both the formation and repair of DNA damage are significantly modulated by chromatin. Our understanding of the impact of chromatin on damage and repair has been significantly enhanced by recent studies. We focus on the nucleosome, the primary building block of chromatin, and discuss how the intrinsic structural properties of nucleosomes, and their associated epigenetic modifications, affect damage formation and DNA repair, as well as subsequent mutagenesis in cancer.     

DNA damage processing at telomeres: The ends justify the means

Telomeres at chromosome ends are nucleoprotein structures consisting of tandem TTAGGG repeats and a complex of proteins termed shelterin. DNA damage and repair at telomeres is uniquely influenced by the ability of telomeric DNA to form alternate structures including loops and G-quadruplexes, coupled with the ability of shelterin proteins to interact with and regulate enzymes in every known DNA repair pathway. The role of shelterin proteins in preventing telomeric ends from being falsely recognized and processed as DNA double strand breaks is well established. Here we focus instead on recent developments in understanding the roles of shelterin proteins and telomeric DNA sequence and structure in processing genuine damage at telomeres induced by endogenous and exogenous DNA damage agents. We will highlight advances in double strand break repair, base excision repair and nucleotide excision repair at telomeres, and will discuss important questions remaining in the field.     

DNA Repair Functions in Heterologous Cells

Abstract

Our genetic information is constantly challenged by exposure to endogenous and exogenous DNA-damaging agents, by DNA polymerase errors, and thereby inherent instability of the DNA molecule itself. The integrity of our genetic information is maintained by numerous DNA repair pathways, and the importance of these pathways is underscored by their remarkable structural and functional conservation across the evolutionary spectrum. Because of the highly conserved nature of DNA repair, the enzymes involved in this crucial function are often able to function in heterologous cells; as an example, the E. coli Ada DNA repair methyltransferase functions efficiently in yeast, in cultured rodent and human cells, in transgenic mice, and in ex vivo-modified mouse bone marrow cells. The heterologous expression of DNA repair functions has not only been used as a powerful cloning strategy, but also for the exploration of the biological and biochemical features of numerous enzymes involved in DNA repair pathways. In this review we highlight examples where the expression of DNA repair enzymes in heterologous cells was used to address fundamental questions about DNA repair processes in many different organisms.     

Poxvirus uracil‐DNA glycosylase—An unusual member of the family I uracil‐DNA glycosylases

Uracil‐DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil‐DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non‐enzymatic function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus‐specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA‐binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. The adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three‐dimensional structure of D4. An overview of the current state of the knowledge on the structure‐function relationship of D4 is provided here.     

Mono- and Bifunctional DNA Glycosylases Involved in Repairing Oxidatively Damaged DNA

DNA repair is a basic biological process providing for the stability and integrity of the genome. Disturbed repair results in premature aging, autoimmune and cardiological disorders, tumorigenesis, etc. Data on enzymes which play key roles in repairing DNA with lesions generated by reactive oxygen species are reviewed. The substrate specificity, mechanism of catalysis, structure of the active center, and specific structural and functional features are described for Escherichia coli mono- and bifunctional DNA glycosylases (endonuclease III, Fpg, MutY, endonuclease VIII, AlkA, MutT) and their prokaryotic and eukaryotic homologs (Ntg1, Ntg2, yOgg1, yOgg2, hOgg1, hOgg2, mOgg1, rOgg1, hMTH, hMYH, MAG, ADPG, and ANPG) which are involved in base excision repair.     

Structural, thermodynamic, and kinetic basis for the activities of some nucleic acid repair enzymes

X-ray structural analysis provides no quantitative estimate of the relative contribution of specific and nonspecific or strong and weak interactions to the total affinity of enzymes for nucleic acids. We have shown that the interaction between enzymes and long nucleic acids at the molecular level can be successfully analyzed by the method of stepwise increase in ligand complexity (SILC). In the present review we summarize our studies of human uracil DNA glycosylase and apurinic/apyrimidinic endonuclease, E. coli 8-oxoguanine DNA glycosylase and RecA protein using the SILC approach. The relative contribution of structural (X-ray analysis data), thermodynamic, and catalytic factors to the discrimination of specific and nonspecific DNA by these enzymes at the stages of complex formation, the following changes in DNA and enzyme conformations and especially the catalysis of the reactions is discussed.     

DNA base repair – recognition and initiation of catalysis

Endogenous DNA damage induced by hydrolysis, reactive oxygen species and alkylation modifies DNA bases and the structure of the DNA duplex. Numerous mechanisms have evolved to protect cells from these deleterious effects. Base excision repair is the major pathway for removing base lesions. However, several mechanisms of direct base damage reversal, involving enzymes such as transferases, photolyases and oxidative demethylases, are specialized to remove certain types of photoproducts and alkylated bases. Mismatch excision repair corrects for misincorporation of bases by replicative DNA polymerases. The determination of the 3D structure and visualization of DNA repair proteins and their interactions with damaged DNA have considerably aided our understanding of the molecular basis for DNA base lesion repair and genome stability. Here, we review the structural biochemistry of base lesion recognition and initiation of one-step direct reversal (DR) of damage as well as the multistep pathways of base excision repair (BER), nucleotide incision repair (NIR) and mismatch repair (MMR).