Balb/c小鼠增龄过程中不同脏器线粒体DNA损伤及损伤修复相关基因的变化

为探讨Balb/c小鼠增龄过程中线粒体DNA损伤及其修复基因表达与衰老之间的关系,采用逆转录 多聚酶链反应（RT PCR）方法,检测年轻与老年Balb/c小鼠脑、肝脏和脾脏中线粒体自身编码基因细胞色素氧化酶亚单位Ⅰ基因（coⅠ）和细胞色素氧化酶亚单位Ⅲ基因（coⅢ）及8 氧鸟嘌呤糖基化酶基因（ogg1）、DNA聚合酶γ（DNA polymeraseγ）基因、胸腺嘧啶乙二醇DNA糖基化酶基因（nth1）等碱基切除修复基因在mRNA水平的变化.用Western印迹方法检测小鼠脾脏中COⅢ和OGG1的蛋白质水平的变化.结果发现,老年小鼠脾脏中coⅠ和coⅢ的mRNA水平比年轻小鼠显著增加（P<0.05）,CO Ⅲ的蛋白质水平亦比年轻小鼠显著升高（P<0.05）;老年小鼠脾脏OGG1的mRNA和蛋白水平上均比年轻小鼠显著增加（P<0.05）.老年小鼠肝脏和脾脏DNA聚合酶γ和NTH1的mRNA水平比年轻小鼠显著升高（P<0.05）.提示线粒体DNA自身编码的基因及碱基切除修复基因的表达失衡可能是Balb/c小鼠衰老的原因之     

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.     

人尿嘧啶糖基化酶cDNA的克隆及其在食管鳞癌组织中的检测

尿嘧啶糖基化酶是碱基切除修复过程的起始酶,对于维护基因稳定具有重要意义。在不同组织及不同细胞周期中,该酶的表达水平存在差异。通过反转录PCR克隆了人尿嘧啶糖基化酶的cDNA编码序列,进一步以克隆所得的已知UNG基因拷贝数的重组质粒作为定量标准,通过实时荧光定量RT-PCR测定了食管癌病人手术切除组织中尿嘧啶糖基化酶的mRNA水平,探讨了尿嘧啶糖基化酶表达水平与食管癌之间的联系。     

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．     

尿嘧啶N糖基化酶(UNG)的研究进展

尿嘧啶N糖基化酶是碱基切除修复过程中的重要组分。本文从酶的概况、在生物体内的分布范围、人尿嘧啶N糖基化酶的基因结构、基因表达与调控、酶的作用机制等方面进行了介绍,并讨论了进一步研究的意义与方向。     

基于CRISPR/Cas系统的DNA碱基编辑技术及其在生物医学和农业中的应用

近年来,基于成簇的规律间隔短回文重复序列及其相关系统(Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein,CRISPR/Cas)的基因编辑技术飞速发展,该系统可以利用同源定向重组(Homology directed repair,HDR)来完成其介导的精准编辑,但效率极低,限制了其在农业和生物医学等领域上的推广应用。基于CRISPR/Cas系统的DNA碱基编辑技术作为一种新兴的基因组编辑技术,能在不产生双链断裂的情况下实现碱基的定向突变,相对于CRISPR/Cas介导的HDR编辑具有更高的编辑效率和特异性。目前,已开发出了可将C碱基突变为T碱基的胞嘧啶碱基编辑器(Cytidine base editors,CBE),将A碱基突变为G碱基的腺嘌呤碱基编辑器(Adenine base editors,ABE),以及可实现碱基任意变换和小片段精准插入和缺失的Prime编辑器(Prime editors,PE)。另外,能实现C到G颠换的糖基化酶碱基编辑器(Glycosylase base editors,GBE)以及能同时编辑A和C两种底物的双碱基编辑器也已被开发出来。文中主要综述了几种DNA碱基编辑器的开发历程、研究进展及各自优点和局限性;介绍了DNA碱基编辑技术在生物医学以及农业中的成功应用案例,以期为DNA碱基编辑器的进一步优化和选择应用提供借鉴。     

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。     

单引物PCR法引入定点突变

目的:利用单个突变引物,在含人呼吸道合胞病毒F蛋白基因编码序列的pc DNA3.1(+)-F质粒中,通过单次环形PCR在特定序列位置引入定点突变。方法:以双链环状的pc DNA3.1(+)-F质粒DNA为模板,设计分别含有三种目的突变N70Q,I431N,Q270T的三条单引物,分别进行单次PCR。用甲基化DNA特异的限制性内切酶Dpn I处理PCR产物后转化大肠杆菌DH5α,进行克隆筛选,酶切鉴定和测序分析。结果:酶切鉴定结果和测序结果均符合预期,利用单引物PCR法成功在含人呼吸道合胞病毒F蛋白基因编码序列的pc DNA3.1(+)-F质粒DNA中引入了单碱基突变、两个间隔碱基突变及相邻三碱基突变三种目的突变。结论:单引物PCR法解决了常规定点突变方法中多个PCR反应,程序繁琐及突变效率低等问题,是一种简便、快速、有效的基因工程定点突变新方法。     

极端嗜热古菌核酸内切酶Ⅲ的研究进展

胸腺嘧啶乙二醇(thymine glycol,Tg)是常见的氧化性DNA损伤碱基之一。DNA中的Tg能够分别阻止DNA聚合酶和RNA聚合酶进行DNA复制和转录,导致相应的生物学过程终止,进而会引起细胞的死亡,因此DNA中的Tg需要被修复。核酸内切酶Ⅲ(endonuclease Ⅲ,EndoⅢ)是一种双功能DNA糖苷酶,能够切除DNA中的Tg,从而启动碱基切除修复途径进行修复DNA中的Tg。细菌、古菌和真核生物的基因组序列中均存在有EndoⅢ蛋白的编码基因。目前,源自于细菌和真核生物的EndoⅢ已有较多的研究,而古菌EndoⅢ的研究相对较少。基于目前已有的极端嗜热古菌EndoⅢ的研究报道,本文综述了极端嗜热古菌EndoⅢ的研究进展,并展望了今后的研究方向。     

植物去甲基化酶ROS1的研究进展

DNA甲基化状态是由从头合成的甲基化、维持型甲基化和DNA主动去甲基化动态调控的结果,由不同调节途径靶向各种酶的催化。5-甲基胞嘧啶DNA糖基化酶/裂解酶ROS1(REPRESSOR OF SILENCING1)是一种DNA去甲基化酶,能够通过启动碱基切除修复途径完成DNA主动去甲基化。介绍了植物中DNA主动去甲基化途径中的去甲基化酶和调节因子;ROS1介导的DNA主动去甲基化的途径;DNA主动去甲基化酶ROS1在各种植物不同发育过程中的作用,包括负调控印记基因表达和种子休眠、调控水稻籽粒品质、影响植物气孔发育等。     

A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD

Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily.     

Selective base excision repair of DNA damage by the non‐base‐flipping DNA glycosylase AlkC

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT‐like repeat (HLR) fold. AlkD uses a unique non‐base‐flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3‐methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non‐base‐flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin‐like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3‐methylcytosine (3mC) and N1‐methyladenine (1mA), which are also repaired by AlkB‐catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.     

A New Family of HEAT-Like Repeat Proteins Lacking a Critical Substrate Recognition Motif Present in Related DNA Glycosylases

DNA glycosylases are important repair enzymes that eliminate a diverse array of aberrant nucleobases from the genomes of all organisms. Individual bacterial species often contain multiple paralogs of a particular glycosylase, yet the molecular and functional distinctions between these paralogs are not well understood. The recently discovered HEAT-like repeat (HLR) DNA glycosylases are distributed across all domains of life and are distinct in their specificity for cationic alkylpurines and mechanism of damage recognition. Here, we describe a number of phylogenetically diverse bacterial species with two orthologs of the HLR DNA glycosylase AlkD. One ortholog, which we designate AlkD2, is substantially less conserved. The crystal structure of Streptococcus mutans AlkD2 is remarkably similar to AlkD but lacks the only helix present in AlkD that penetrates the DNA minor groove. We show that AlkD2 possesses only weak DNA binding affinity and lacks alkylpurine excision activity. Mutational analysis of residues along this DNA binding helix in AlkD substantially reduced binding affinity for damaged DNA, for the first time revealing the importance of this structural motif for damage recognition by HLR glycosylases.     

A new protein architecture for processing alkylation damaged DNA: the crystal structure of DNA glycosylase AlkD

DNA glycosylases safeguard the genome by locating and excising chemically modified bases from DNA. AlkD is a recently discovered bacterial DNA glycosylase that removes positively charged methylpurines from DNA, and was predicted to adopt a protein fold distinct from from those of other DNA repair proteins. The crystal structure of Bacillus cereus AlkD presented here shows that the protein is composed exclusively of helical HEAT-like repeats, which form a solenoid perfectly shaped to accommodate a DNA duplex on the concave surface. Structural analysis of the variant HEAT repeats in AlkD provides a rationale for how this protein scaffolding motif has been modified to bind DNA. We report 7mG excision and DNA binding activities of AlkD mutants, along with a comparison of alkylpurine DNA glycosylase structures. Together, these data provide important insight into the requirements for alkylation repair within DNA and suggest that AlkD utilizes a novel strategy to manipulate DNA in its search for alkylpurine bases.     

Alkylpurine Glycosylase D Employs DNA Sculpting as a Strategy to Extrude and Excise Damaged Bases

Alkylpurine glycosylase D (AlkD) exhibits a unique base excision strategy. Instead of interacting directly with the lesion, the enzyme engages the non-lesion DNA strand. AlkD induces flipping of the alkylated and opposing base accompanied by DNA stack compression. Since this strategy leaves the alkylated base solvent exposed, the means to achieve enzymatic cleavage had remained unclear. We determined a minimum energy path for flipping out a 3-methyl adenine by AlkD and computed a potential of mean force along this path to delineate the energetics of base extrusion. We show that AlkD acts as a scaffold to stabilize three distinct DNA conformations, including the final extruded state. These states are almost equivalent in free energy and separated by low barriers. Thus, AlkD acts by sculpting the global DNA conformation to achieve lesion expulsion from DNA. N-glycosidic bond scission is then facilitated by a backbone phosphate group proximal to the alkylated base.     

The substrate binding interface of alkylpurine DNA glycosylase AlkD

Tandem helical repeats have emerged as an important DNA binding architecture. DNA glycosylase AlkD, which excises N3- and N7-alkylated nucleobases, uses repeating helical motifs to bind duplex DNA and to selectively pause at non-Watson–Crick base pairs. Remodeling of the DNA backbone promotes nucleotide flipping of the lesion and the complementary base into the solvent and toward the protein surface, respectively. The important features of this new DNA binding architecture that allow AlkD to distinguish between damaged and normal DNA without contacting the lesion are poorly understood. Here, we show through extensive mutational analysis that DNA binding and N3-methyladenine (3mA) and N7-methylguanine (7mG) excision are dependent upon each residue lining the DNA binding interface. Disrupting electrostatic or hydrophobic interactions with the DNA backbone substantially reduced binding affinity and catalytic activity. These results demonstrate that residues seemingly only involved in general DNA binding are important for catalytic activity and imply that base excision is driven by binding energy provided by the entire substrate interface of this novel DNA binding architecture.     

Nucleic acid recognition by tandem helical repeats

Protein domains constructed from tandem α-helical repeats have until recently been primarily associated with protein scaffolds or RNA recognition. Recent crystal structures of human mitochondrial termination factor MTERF1 and Bacillus cereus alkylpurine DNA glycosylase AlkD bound to DNA revealed two new superhelical tandem repeat architectures capable of wrapping around the double helix in unique ways. Unlike DNA sequence recognition motifs that rely mainly on major groove read-out, MTERF and ALK motifs locate target sequences and aberrant nucleotides within DNA by resculpting the double-helix through extensive backbone contacts. Comparisons between MTERF and ALK repeats, together with recent advances in ssRNA recognition by Pumilio/FBF (PUF) domains, provide new insights into the fundamental principles of protein-nucleic acid recognition.     

Structural insight into repair of alkylated DNA by a new superfamily of DNA glycosylases comprising HEAT-like repeats

3-methyladenine DNA glycosylases initiate repair of cytotoxic and promutagenic alkylated bases in DNA. We demonstrate by comparative modelling that Bacillus cereus AlkD belongs to a new, fifth, structural superfamily of DNA glycosylases with an alpha-alpha superhelix fold comprising six HEAT-like repeats. The structure reveals a wide, positively charged groove, including a putative base recognition pocket. This groove appears to be suitable for the accommodation of double-stranded DNA with a flipped-out alkylated base. Site-specific mutagenesis within the recognition pocket identified several residues essential for enzyme activity. The results suggest that the aromatic side chain of a tryptophan residue recognizes electron-deficient alkylated bases through stacking interactions, while an interacting aspartate-arginine pair is essential for removal of the damaged base. A structural model of AlkD bound to DNA with a flipped-out purine moiety gives insight into the catalytic machinery for this new class of DNA glycosylases.     

Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA

Human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC), a key oxidation product of 5-methylcytosine in genomic DNA, in a recently discovered cytosine demethylation pathway. We present here the crystal structures of the hTDG catalytic domain in complex with duplex DNA containing either 5caC or a fluorinated analog. These structures, together with biochemical and computational analyses, reveal that 5caC is specifically recognized in the active site of hTDG, supporting the role of TDG in mammalian 5-methylcytosine demethylation.