紫外诱导植物产生DNA损伤的修复机制

日光中的紫外线可以诱导生物体的DNA产生损伤,产生的损伤主要有两种：环丁烷嘧啶二聚体（CPD）和6-4光产物（即6-4嘧啶二聚体）.这些损伤如果不经修复则可能会导致生物体死亡.最近的研究证明,植物可以通过多种途径来修复紫外诱导的DNA损伤,包括6-4光产物和CPD的光修复作用.此外,植物还可以通过一般的核酸切除修复（NER）以及旁路聚合酶（bypass polymerase）来修复损伤.     

T4核酸内切酶V的分离纯化

 <正> T_4核酸内切酶V[endodeoxyribonuclease(Pyrimidine dimer)EC 3.1.25.1,简称Endo V]是由噬菌体T_4感染宿主E.coli后产生的一种修复酶。它能特异识别紫外线(UV)辐射在DNA引起的损伤产物环丁烷嘧啶二聚体(PD),在组成PD的两个嘧啶核苷酸之间切割磷酸二酯键,造成单链缺刻,从而启动E.coli体内的切除修复途径~[1]。Endo V不仅具有对PD的高     

UV—A区段紫外线照射对DNA影响的拉曼光谱分析

本文检测了鲱鱼精DNA水溶液经不同时间UV－A紫外辐射后的拉曼光谱,研究结果表明,该区段紫外辐射比用UV－A和UV－B共同照射对DNA的影响要小,主链构象基本稳定。但经较长时间辐射仍会对鲱鱼精DNA造成损伤,受影响的部位主要是脱氧核糖和胸腺嘧啶碱基部分,UV－A对脱氧核糖的影响与UV－A加UV－B共同照射的结果作比较后,可以说明UV－A对脱氧核糖的损伤有累积的效应,而对于胸腺嘧啶的影响,从其各个指标的分析来看,有损伤但程度较小。本实验说明UV－A辐射条件下没有嘧啶二聚体的形成,也不存在6,4光产物形成的证明,但对于Dewar异构体的形成,有部分证明,与Taylor(1994)报道的结果相一致,UV－A没有造成DNA单链断裂现象。     

紫外线B区对小牛胸腺DNA损伤的拉曼光谱研究

用拉曼光谱检测了远紫外区UVB(280m～320nm)对小牛胸腺DNA的损伤,并对这个区段紫外线对DNA的不同照射时间时的损伤特征进行了比较分析。实验中所用的紫外线强度与太阳光强度相当。结果表明,辐照3h以内时,UVB对DNA的构型有较明显的损伤,这可能是受到嘧啶碱基损伤的影响。相对而言,UVB对脱氧核糖和碱基的损伤要严厉得多。就碱基对的受损伤程度来说,嘧啶碱受损最严重,部分证明了环丁烷嘧啶二聚体和6-4光产物的形成。经过3h UVB照射,AT碱基对和和胞嘧啶环的堆叠程度有所瓦解,一些碱基对受到修饰。而一些拉曼特征峰强度的反复波动,则说明了长时间的紫外照射可以导致部分DNA光复活的产生。进一步分析发现,UVB以一种较快的方式对DNA产生损伤。     

DNA光解酶的结构与功能研究进展

对近年来在包括酶蛋白、辅酶的DNA光解酶结构及其与功能关系方面的研究进展作了综述.DNA光解酶可通过光诱导的电子转移催化裂解环丁烷嘧啶二聚体(Pyr<>Pyr),从而修复紫外线引起的DNA的主要损伤.研究发现,来自不同有机体的光解酶均含有两个非共价的辅基一个是1,5-二氢黄素腺嘌呤二核苷酸(FADH2),另一个是次甲基四氢叶酸(MTHF)或8-羟基-5-去氮杂核黄素(8-HDF),前者具有催化活性,可在光作用下通过电子转移裂解嘧啶二聚体,后者不具有催化活性,但能收集光子并将能量传递给FADH2,具有“天线”作用.     

DNA修复酶的结构和功能

DNA修复酶是一类能保护生物体免受各种DNA损伤的毒性效应和保证遗传信息完整性的重要酶蛋白。近年来对DNA修复酶晶体结构的研究揭示了一些结构基序参与了酶蛋白与特定DNA损伤的识别过程,这些研究结果促进了对修复特定DNA损伤的作用机理和结构基础的认识和了解。本文综述了这方面的研究进展。     

温度对UV-B诱导的烟草叶圆片DNA损伤的影响

环丁烷嘧啶二聚体(CPD)和6-4光产物(6-4PP)是两种主要的UV-B诱导的DNA光损伤产物。利用单克隆抗体酶联免疫吸附分析法(ELISA),研究了温度对UV-B诱导的烟草叶圆片DNA损伤的影响。室温(24℃)条件下,UV-B处理引起了烟草叶圆片DNA中CPD和6-4PP的积累。0℃条件下,UV-B处理的烟草叶圆片DNA中CPD和6-4PP的积累比室温下分别降低了9.8%和12%。UV-B诱导的DNA损伤曾被认为是纯粹的光化学过程而与不受温度影响,而本实验结果表明,UV-B诱导的烟草叶圆片DNA形成CPD和6-4PP的过程具有温度依赖性。这一特性有利于植物对全球变化的适应,因而具有重要的生态学意义。     

核苷酸切除修复对UVB光损伤的识别机制

中波紫外线（UVB）会对皮肤造成各种损伤,这些都根源于UVB对皮肤细胞DNA的光损伤。光损伤产物主要有环丁烷嘧啶二聚体（CPD）和64光产物（6-4PP）两类,还包括少量的氧化损伤。CPD和6-4PP的修复是由核苷酸切除修复（NER）执行的。NER可分为全基因组核苷酸切除修复（GGR）和转录耦联核苷酸切除修复（TCR）两个亚途径。识别因子XPC通过一种不直接识别损伤本身的机制在GGR识别过程中发挥作用;在TCR识别过程中强调了关键因子CSB单体及二聚体两种形式的转换。在染色质水平上,DDB介导的泛素化作用是NER识别过程中重要的调控要素。另外,完成使命的识别因子的最终走向也是NER途径中的一个重要环节。通过分析上述生化过程,较清楚地总结了GGR及TCR对UVB导致的光损伤的识别机制。     

紫外线为何能杀死病菌？

紫外线为何能杀死病菌？答：紫外线照射引起病菌细胞的核酸ＤＮＡ链的磷酸二脂键和氢键断裂,胸腺嘧啶（Ｔ）与胞嘧啶（Ｃ）的水合作用以及胸腺嘧啶的二聚体（ＴＴ）形成,这样便引起ＤＮＡ双链结构扭曲变形,阻碍碱基间的正常配对,破坏ＤＮＡ模板的正常功能,影响双链的...     

用病毒探针研究紫外线诱导的DNA损伤的致死效应

紫外线(254nm)能诱导一系列DNA损伤。嘧啶二聚体是至今所知的主要损伤产物。然而,由于细胞基因组结构和复制方式的复杂性以及DNA损伤修复途径的多样性,定量地研究特定DNA损伤的生物学效应是困难的。微小病毒是一种良好的探针,因它们有特有的基因组结构和复制方式,它们已被成功地用于探测细胞的诱发性易错修复功能和突变过程。在此项研究中,自主性微小病毒     

Role of human DNA polymerase κ in extension opposite from a cis-syn thymine dimer

Exposure of DNA to UV radiation causes covalent linkages between adjacent pyrimidines. The most common lesion found in DNA from these UV-induced linkages is the cis-syn cyclobutane pyrimidine dimer. Human DNA polymerase κ (Polκ), a member of the Y-family of DNA polymerases, is unable to insert nucleotides opposite the 3'T of a cis-syn T-T dimer, but it can efficiently extend from a nucleotide inserted opposite the 3'T of the dimer by another DNA polymerase. We present here the structure of human Polκ in the act of inserting a nucleotide opposite the 5'T of the cis-syn T-T dimer. The structure reveals a constrained active-site cleft that is unable to accommodate the 3'T of a cis-syn T-T dimer but is remarkably well adapted to accommodate the 5'T via Watson-Crick base pairing, in accord with a proposed role for Polκ in the extension reaction opposite from cyclobutane pyrimidine dimers in vivo.     

Efficiency of pyrimidine dimer formation in Escherichia coli across UV wavelengths

AIMS: Inactivation of Escherichia coli as a function of ultraviolet (UV) wavelength was investigated by using the endonuclease-sensitive site (ESS) assay to quantify pyrimidine dimer formation. METHODS AND RESULTS: Ultraviolet dose-response curves were determined based on both log reduction in colony-forming units (CFU) and endonuclease-sensitive sites per kb DNA (ESS/kb) for monochromatic 254-nm low-pressure (LP) UV, polychromatic medium-pressure (MP) UV, 228 and 289-nm UV irradiation. UV irradiation from LP and MP UV sources were approx. equal in both CFU reduction and pyrimidine dimer formation at all UV doses studied; 228-nm irradiation was less effective than LP or MP, and 289-nm irradiation was the least effective in both CFU reduction and pyrimidine dimer formation. These results are in qualitative agreement with the absorption spectrum of pyrimidine bases in DNA. Results indicated an approx. linear relationship between ESS/kb and log CFU reduction. CONCLUSIONS: Formation of pyrimidine dimers in genomic DNA is primarily responsible for UV inactivation of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributed to fundamental understanding of UV disinfection and aids in UV reactor design.     

Structure of T4 pyrimidine dimer glycosylase in a reduced imine covalent complex with abasic site-containing DNA

The base excision repair (BER) pathway for ultraviolet light (UV)-induced cyclobutane pyrimidine dimers is initiated by DNA glycosylases that also possess abasic (AP) site lyase activity. The prototypical enzyme known to catalyze these reactions is the T4 pyrimidine dimer glycosylase (T4-Pdg). The fundamental chemical reactions and the critical amino acids that lead to both glycosyl and phosphodiester bond scission are known. Catalysis proceeds via a protonated imine covalent intermediate between the alpha-amino group of the N-terminal threonine residue and the C1' of the deoxyribose sugar of the 5' pyrimidine at the dimer site. This covalent complex can be trapped as an irreversible, reduced cross-linked DNA-protein complex by incubation with a strong reducing agent. This active site trapping reaction is equally efficient on DNA substrates containing pyrimidine dimers or AP sites. Herein, we report the co-crystal structure of T4-Pdg as a reduced covalent complex with an AP site-containing duplex oligodeoxynucleotide. This high-resolution structure reveals essential precatalytic and catalytic features, including flipping of the nucleotide opposite the AP site, a sharp kink (approximately 66 degrees ) in the DNA at the dimer site and the covalent bond linking the enzyme to the DNA. Superposition of this structure with a previously published co-crystal structure of a catalytically incompetent mutant of T4-Pdg with cyclobutane dimer-containing DNA reveals new insights into the structural requirements and the mechanisms involved in DNA bending, nucleotide flipping and catalytic reaction.     

Assay of DNA Photolyase Activity in Spinach Leaves in Relation to Cell Compartmentation-Evidence for Lack of DNA Photolyase in Chloroplasts

Spinach cyclobutane pyrimidine dimer (CPD)-specific DNA photolyase was successfully detected in leaf extracts by an assay system for plant photolyase using an improved enzyme-linked immunosorbent assay (ELISA) which was newly introduced by novel horseradish peroxidase (HRP)-linked CPD specific monoclonal antibodies. The assay system includes two main steps: a photorepair reaction of CPD introduced in substrate DNA and measurement of CPD remained after the photorepair by the improved ELISA. When CPD- induced salmon sperm DNA was used as a substrate, high CPD-photolyase activities were observed in the enzyme fraction prepared from whole spinach leaf extracts, but not from chloroplast extracts. This strongly suggests that spinach CPD-specific photolyases are localized in cell compartments other than chloroplasts.     

Thermodynamics and kinetics for base pair opening in the DNA decamer duplexes containing cyclobutane pyrimidine dimer

The cyclobutane pyrimidine dimer (CPD) is one of the major classes of cytotoxic and carcinogenic DNA photoproducts induced by UV light. Hydrogen exchange rates of the imino protons were measured for various CPD-containing DNA duplexes to better understand the mechanism for CPD recognition by XPC-hHR23B. The results here revealed that double T·G mismatches in a CPD lesion significantly destabilized six consecutive base pairs compared to other DNA duplexes. This flexibility in a DNA duplex caused at the CPD lesions with double T·G mismatches might be the key factor for damage recognition by XPC-hHR23B.     

Mutability of bacteriophage M13 by ultraviolet light: Role of pyrimidine dimers

Summary The role of pyrimidine dimers in mutagenesis by ultraviolet light was examined by measuring the UV-induced reversion of six different bacteriophage M13 amber mutants for which the neighboring DNA sequences are known. The mutational response at amber (TAG) codons preceded by a guanine or adenine (where no pyrimidine dimer can be formed) were compared with those preceded by thymine or cytosine (where dimer formation is possible). Equivalent levels of UV-induced mutagenesis were observed at both kinds of sites. This observation demonstrates that there is no requirement for a pyrimidine dimer directly at the site of UV-induced mutation in this single-stranded DNA phage. UV irradiation of the phage was also performed in the presence of Ag⁺ ions, which specifically sensitize the DNA to dimer formation. The two methods of irradiation, when compared at equal survival levels (and presumably equal dimer frequencies), produced equivalent frequencies of reversion of the amber phage. We believe these results indicate that while the presence of pyrimidine dimers may be a prerequisite for UV mutagenesis, the actual mutagenic event can occur at a site some distance removed from a dimer.     

Utilization of DNA photolyase, pyrimidine dimer endonucleases, and alkali hydrolysis in the analysis of aberrant ABC excinuclease incisions adjacent to UV-induced DNA photoproducts.

ABC excinuclease of Escherichia coli removes 6-4 photoproducts and pyrimidine dimers from DNA by making two single strand incisions, one 8 phosphodiester bonds 5' and another 4 or 5 phosphodiester bonds 3' to the lesion. We describe in this communication a method, which utilizes DNA photolyase from E. coli, pyrimidine dimer endonucleases from M. luteus and bacteriophage T4, and alkali hydrolysis, for analyzing the ABC excinuclease incision pattern corresponding to each of these photoproducts in a DNA fragment. On occasion, ABC excinuclease does not incise DNA exclusively 8 phosphodiester bonds 5' or 4 or 5 phosphodiester bonds 3' to the photoproduct. Both the nature of the adduct (6-4 photoproduct or pyrimidine dimer) and the sequence of neighboring nucleotides influence the incision pattern of ABC excinuclease. We show directly that photolyase stimulates the removal of pyrimidine dimers (but not 6-4 photoproducts) by the excinuclease. Also, photolyase does not repair CC pyrimidine dimers efficiently while it does repair TT or TC pyrimidine dimers.     

Antigen structural requirements for recognition by a cyclobutane thymine dimer-specific monoclonal antibody.

A monoclonal antibody (TDM-2) specific to a UV-induced cyclobutane pyrimidine dimer (T[cis-syn]T) has previously been established; however,the immunization had used UV-irradiated calf-thymus DNA containing a heterogeneous mixture of photoproduct sites. We investigated here the structural requirements of antigen recognition by the antibody using chemically synthesized antigen analogs. TDM-2 bound with cis-syn,but not trans-syn thymine dimer,and could bind strongly with four nucleotide analogs in which the cis-syn pyrimidine dimer was located in the center. Antigen analogs containing abasic linkers at the 5'- or 3'-side of the cis-syn cyclobutane pyrimidine dimer were synthesized and tested for binding to TDM-2. The results indicated that TDM-2 recognizes not only the cyclobutane ring but also both the 5'- and 3'-side nucleosides of the cyclobutane dimer. Furthermore,it was proved that either the 5'- or 3'-side phosphate group at a cyclobutane dimer site was absolutely required for the affinity to TDM-2. The antibody showed a strong binding to single stranded DNA but indicated little binding to double stranded DNA.     

Increased DNA repair in <Emphasis Type="Italic">Arabidopsis</Emphasis> plants overexpressing CPD photolyase

Ultraviolet-B (UV-B, 280–320 nm) radiation may have severe negative effects on plants including damage to their genetic information. UV protection and DNA-repair mechanisms have evolved to either avoid or repair such damage. Since autotrophic plants are dependent on sunlight for their energy supply, an increase in the amount of UV-B reaching the earth’s surface may affect the integrity of their genetic information if DNA damage is not repaired efficiently and rapidly. Here we show that overexpression of cyclobutane pyrimidine dimer (CPD) photolyase (EC 4.1.99.3) in Arabidopsis thaliana (L.), which catalyses the reversion of the major UV-B photoproduct in DNA (CPDs), strongly enhances the repair of CPDs and results in a moderate increase of biomass production under elevated UV-B.     

The relationship between pyrimidine dimers and replicating DNA in UV-irradiated human fibroblasts.

The relationship between pyrimidine dimers (measured as endonuclease-sensitive sites) and newly-synthesized DNA has been examined in several different ways, with the following results:- 1. After UV-irradiation of normal human fibroblasts the frequency of pyrimidine dimer sites in sections of DNA which have been synthesized immediately before the UV-irradiation is similar to that in the bulk DNA. 2. The frequency of pyrimidine dimer sites in the parental strands of replicating DNA in UV-irradiated normal human fibroblasts is similar to that in the bulk DNA. 3. In UV-irradiated XP variant cells the size of DNA synthesized in the presence of caffeine immediately after UV irradiation accurately corresponds with the average interdimer distance in the parental DNA. This suggests that in this experimental situation each pyrimidine dimer gives rise to a disocntinuity or a termination site in the daughter strand.