DNA修复:为生命提供化学稳定性——2015年诺贝尔化学奖解读

托马斯·林达尔(Tomas Lindahl)、保罗·莫德里奇(Paul Modrich)以及阿齐兹·桑贾尔(Aziz Sancar)因为从分子水平上解释了细胞进行DNA损伤修复的机制,被授予2015年诺贝尔化学奖。3位科学家的研究成果,解释了活体细胞DNA修复的运作机制,为治疗癌症这一人类的顽疾奠定了基础。     

顺铂耐药的分子机制

顺铂被广泛用于多种类型的实体肿瘤的临床治疗.DNA是顺铂的主要靶点,顺铂结合会导致DNA损伤并诱发细胞凋亡.然而,顺铂化疗常常受到内在的和获得性的耐药性的限制.在过去30多年里,大量的研究致力于对顺铂耐药性的理解,并且提出了几种导致顺铂耐药性的分子机制.这些机制显示顺铂的耐药性具有多因素特征.本文系统描述和讨论了顺铂的耐药性机制,包括细胞内药物积累的减少,药物去活作用的增强,DNA修复作用,DNA损伤反应和凋亡通路的变化以及一些间接信号通路的调控影响.     

DNA脱碱基位点的检测方法及其生物学研究进展

DNA中糖苷键断裂形成的脱碱基位点（脱嘌呤/嘧啶位点,AP位点）是常见的DNA损伤类型之一,由核苷酸自发水解产生,也是DNA碱基切除修复途径中的关键中间体。若修复不及时,可能会导致DNA复制阻滞和DNA链断裂,产生突变和细胞毒性,同时还会引起形成DNA交联或DNA-蛋白质交联的损伤,因此对于AP损伤的检测有助于理解细胞氧化应激损伤和基因毒性物质的毒性评价。目前检测AP位点的方法有14C或32P后标记法、酶联免疫吸附分析（ELISA）法和液相色谱-质谱（LC-MS）技术等。该文重点概述DNA中AP位点的检测方法和生物学研究进展,并展望了AP位点损伤相关研究的前景。     

自修复高分子材料近五年的研究进展

自修复材料的概念源于对生物体自愈合现象的仿生研究,该类材料在受到损伤时可进行自修复并恢复一定程度的力学等性能。对高分子材料而言,其受机械力损伤后一般发生大分子链均裂或异裂而使材料产生微裂纹,此类微裂纹很难探测,而微裂纹的产生往往会引起高分子材料失效,因此快速修复微裂纹对诸多工程领域的高分子材料来讲尤为重要。本文从外源型及本征型自修复高分子材料两个方面,综述了近五年自修复高分子材料的研究进展,并对其今后发展进行了展望。     

结构用自修复型高分子材料的制备

自修复型高分子材料属于智能材料的一类,仿照生物体损伤自愈合的功能,通过材料内部的自诊断和自响应机制,及时修复材料在成型加工或使用过程产生的微小裂纹,避免其进一步扩展.近年来本课题组针对结构用自修复型高分子材料的强度恢复问题,综合利用高分子化学、高分子物理、材料力学等学科的理论和方法,设计、合成了一系列外植型和本征型自修复高分子材料,提出的自修复策略适用于典型热固性和热塑性高分子材料.此外,深入研究了相关的合成路线、配方优化、制备工艺、材料结构与性能、自修复的微观机制、使用稳定性等,为此类材料的实际应用提供依据.     

DNA光复活作用机理的研究进展*

"环丁烷型嘧啶二聚体(Pyr< > Pyr) 是太阳光中紫外线造成DNA 损伤的主要光化学产物。DNA 光复活酶(或称光解酶) 能够利用可见光裂解二聚体的环丁烷环而修复DNA。本文对DNA 光复活过程中的光解酶对Pyr< > Pyr 的识别和光催化Pyr< > Pyr 裂解反应进行了综述, 介绍了DNA 光解酶的结构、DNA 的主要UV 光化学产物。较详尽地评述了国际上在光解酶催化二聚体裂解的途径以及模型研究方面的最新进展, 并预测了该领域的发展前景。     

科学家发现金属材料自我修复机制

自然界中的生物体和具有记忆功能的有机材料等,在遭受损伤时具有自我康复的功能。麻省理工学院的研究人员在一项金属特性实验中意外发现受损的金属也具有自我修复的功能。金属合金分子结构电脑模拟显示,微晶粒之间的边界会在压力下出现裂痕。大多数金属都是由细微的晶粒构成,这些晶粒的大小和方向能够影响金属的强度和特性。但在某些条件下,压力可以让这种晶粒的微观结构发生改变:使晶界(晶粒边界)发生移动,而晶界移动则是修复"创伤"的关键。     

错配核酸识别修复的研究进展*

综述了当前发展起来的识别修复错配核酸的化学模型的最新进展。此项研究会对阐明生物体内DNA的识别修复机理、合理设计新的人工核酸酶提供理论指导。     

锂离子电池自修复聚合物电解质的研究进展

自修复材料可以修复其在外界环境因素作用下产生的局部创伤或微裂纹,大大延长材料的使用寿命.将自修复聚合物作为固态聚合物电解质应用于锂离子电池,可以显著提高锂离子电池的循环稳定性和安全性,延长使用寿命.本文首先概述了自修复聚合物材料的发展历程和修复机理,然后按超分子相互作用和动态共价键分类总结了本征型自修复聚合物电解质应用于锂离子电池的研究进展,最后对自修复聚合物电解质存在的问题和未来的发展方向做出了展望,为下一代高安全性、高性能和长使用寿命的锂离子电池的研究提供借鉴.     

致癌性卤代醌类消毒副产物造成 DNA 损伤的分子机理研究

卤代醌是一类卤代芳烃类环境污染物的致癌中间体,也是在饮用水中新发现的氯化消毒副产物。我们最近发现卤代醌和 H₂O₂ 或有机氢过氧化物体系可以不依赖过渡金属离子,而产生高活性的羟基/烷氧自由基和醌氧/醌碳自由基。目前尚不清楚这些卤代醌类致癌物和氢过氧化物共存能否诱导 DNA 产生氧化损伤和修饰,以及其潜在的分子机制是什么。我们的研究发现 DNA 在四氯-1,4-苯醌/H₂O₂体系中可被氧化产生 8-氧脱氧鸟苷、DNA 链断裂和三种甲基氧化产物,这些反应不依赖过渡金属离子,且由于卤代醌与 DNA 的嵌入作用而导致其氧化作用增强。其他卤代醌也观察到了类似的现象,而且通常比经典的 Fenton 体系更有效。我们进一步将研究从纯化的 DNA 扩展到了活细胞的基因组 DNA。同时还发现卤代醌和有机氢过氧化物(如叔丁基过氧化氢或在正常生理条件下产生的 13S-过氧羟基-9Z,11E-十八碳二烯酸(13-HPODE))共存时,可通过独特的醌氧自由基介导机制诱导 DNA 氧化生成致突变性更强的咪唑啉酮类产物 dIz。这些发现为解释普遍存在的卤代醌类致癌中间体和消毒副产物的潜在基因毒性、致突变性和致癌性提供了新思路。     

Repair processes on deoxyribonucleic acid

Many cells have the ability to recognize and eliminate damage to their DNA, particularly thymine dimers formed by UV light. The elimination of this damage may be achieved by enzymatic, light-dependent cleavage of the dimers into the monomers (photoreactivation) or more frequently by dark repair, in which the damaged part is completely removed from the, DNA. In this repair process, the DNA is incised by an endonuclease in the immediate vicinity of the thymine dimers. Oligonucleotides containing the thymine dimer are removed hydrolytically from the DNA by the 5→3′ exonuclease activity of DNA polymerase I (Kornberg enzyme). The resulting gaps are immediately closed by a de novo synthesis with the aid of the same DNA polymerase I, the complementary strand serving as a template (excision repair). The final step is the formation of the phosphodiester bond between the newly synthesized DNA fragment and the old DNA strand by a DNA ligase. Xeroderma pigmentosum patients lack the endonuclease as a result of a genetic defect; they therefore cannot eliminate thymine dimers from their DNA, and are extremely sensitive to sunlight. All information so far suggests that genetic recombination and DNA repair are performed by the same enzyme system.     

An Excimer Clamp for Measuring Damaged‐Base Excision by the DNA Repair Enzyme NTH1

Direct measurement of DNA repair enzyme activities is important both for the basic study of cellular repair pathways as well as for potential new translational applications in their associated diseases. NTH1, a major glycosylase targeting oxidized pyrimidines, prevents mutations arising from this damage, and the regulation of NTH1 activity is important in resisting oxidative stress and in suppressing tumor formation. Herein, we describe a novel molecular strategy for the direct detection of damaged DNA base excision activity by a ratiometric fluorescence change. This strategy utilizes glycosylase‐induced excimer formation of pyrenes, and modified DNA probes, incorporating two pyrene deoxynucleotides and a damaged base, enable the direct, real‐time detection of NTH1 activity in vitro and in cellular lysates. The probe design was also applied in screening for potential NTH1 inhibitors, leading to the identification of a new small‐molecule inhibitor with sub‐micromolar potency.     

A Platinum(IV) Anticancer Prodrug Targeting Nucleotide Excision Repair To Overcome Cisplatin Resistance

DNA damage response plays a key role not only in maintaining genome integrity but also in mediating the antitumor efficacy of DNA‐damaging antineoplastic drugs. Herein, we report the rational design and evaluation of a Pt^IV anticancer prodrug inhibiting nucleotide excision repair (NER), one of the most pivotal processes after the formation of cisplatin‐induced DNA damage that deactivates the drug and leads to drug resistance in the clinic. This dual‐action prodrug enters cells efficiently and causes DNA damage while simultaneously inhibiting NER to promote apoptotic response. The prodrug is strongly active against the proliferation of cisplatin‐resistant human cancer cells with an up to 88‐fold increase in growth inhibition compared with cisplatin, and the prodrug is much more active than a mixture of cisplatin and an NER inhibitor. Our study highlights the importance of targeting downstream pathways after the formation of Pt‐induced DNA damage as a novel strategy to conquer cisplatin resistance.     

Self‐assembled Lipid Nanoparticles for Ratiometric Codelivery of Cisplatin and siRNA Targeting XPF to Combat Drug Resistance in Lung Cancer

DNA damage repair through the nucleotide excision repair (NER) pathway is one of the major reasons for the decreased antitumor efficacy of platinum‐based anticancer drugs that have been widely applied in the clinic. Inhibiting the intrinsic NER function may enhance the antitumor activity of cisplatin and conquer cisplatin resistance. Herein, we report the design, optimization, and application of a self‐assembled lipid nanoparticle (LNP) system to simultaneously deliver a cisplatin prodrug together with siRNA targeting endonuclease xeroderma pigmentosum group F (XPF), a crucial component in the NER pathway. The LNP is able to efficiently encapsulate both the platinum prodrug and siRNA molecules with a tuned ratio. Both platinum prodrug and XPF‐targeted siRNA are efficiently carried into cells and released; the former damages DNA and the latter specifically downregulates both mRNA and protein levels of XPF to potentiate the platinum drug, leading to enhanced expression levels of apoptosis markers and improved cytotoxicity in both cisplatin‐sensitive and ‐resistant human lung cancer cells. Our results demonstrate an effective approach to utilize a multi‐targeted nanoparticle system that can specifically silence an NER‐related gene to promote apoptosis induced by cisplatin, especially in cisplatin‐refractory tumors.     

On-bead fluorescent DNA nanoprobes to analyze base excision repair activities

DNA integrity is constantly threatened by endogenous and exogenous agents that can modify its physical and chemical structure. Changes in DNA sequence can cause mutations sparked by some genetic diseases or cancers. Organisms have developed efficient defense mechanisms able to specifically repair each kind of lesion (alkylation, oxidation, single or double strand break, mismatch, etc). Here we report the adjustment of an original assay to detect enzymes’ activity of base excision repair (BER), that supports a set of lesions including abasic sites, alkylation, oxidation or deamination products of bases. The biosensor is characterized by a set of fluorescent hairpin-shaped nucleic acid probes supported on magnetic beads, each containing a selective lesion targeting a specific BER enzyme. We have studied the DNA glycosylase alkyl-adenine glycosylase (AAG) and the human AP-endonuclease (APE1) by incorporating within the DNA probe a hypoxanthine lesion or an abasic site analog (tetrahydrofuran), respectively. Enzymatic repair activity induces the formation of a nick in the damaged strand, leading to probe's break, that is detected in the supernatant by fluorescence. The functional assay allows the measurement of DNA repair activities from purified enzymes or in cell-free extracts in a fast, specific, quantitative and sensitive way, using only 1 pmol of probe for a test. We recorded a detection limit of 1 μg mL⁻¹ and 50 μg mL⁻¹ of HeLa nuclear extracts for APE1 and AAG enzymes, respectively. Finally, the on-bead assay should be useful to screen inhibitors of DNA repair activities.     

uvrB-dependent, recF-independent post-replication (or replication) repair in Escherichia coli

In UV-damaged cells, a large fraction of pyrimidine dimers may remain unexcised and may be tolerated by a uvrB recA lexA-dependent non-excisional mode of repair (M. Sedliaková, J. Brozmanová, F. Ma ek and K. Kleibl, Biophys. J., 36 (1981) 429-441). We show here that a similar repair pathway operates in the Escherichia coli recF 143 single mutant but not in the recF uvrB double mutant. This indicates that the putative repair pathway is recF independent.     

The potential roles of p53 tumor suppressor in nucleotide excision repair (NER) and base excision repair (BER)

The p53 tumor suppressor has long been envisaged to preserve genetic stability by the induction of cell cycle checkpoints and apoptosis. More recently, p53 has been implicated to play roles in DNA repair responses to genotoxic stresses. UV-damage and the damage caused by certain chemotherapeutics including cisplatin and nitrogen mustards are known to be repaired by the nucleotide excision repair (NER) pathway which is reportedly regulated by p53 and its downstream genes. There are evidences to suggest that the base excision repair (BER) induced by the base-damaging agent methyl methanesulfonate (MMS) is partially deficient in cells lacking functional p53. This result suggests that the activity of BER might be also dependent on the p53 status. In this review, we discuss the possibilities that p53 regulates BER as well as NER; these are one of the most significant potentials of p53 tumor suppressor for repairing the vast majority of DNA damages that is incurred from various environmental stresses.     

Charge Transfer through the DNA Base Stack

Whether the DNA base pair stack might serve as a medium for efficient, long-range charge transfer has been debated almost since the first proposal of the double-helical structure of DNA. The consequences of long-range radical migration through DNA are important with respect to understanding carcinogenesis and mutagenesis. Double-helical DNA has in its core a stacked array of aromatic heterocyclic base pairs, and this molecular π stack represents a unique system in which to explore the chemistry of electron transfer. We designed a family of metal complexes which bind to DNA by intercalative stacking within the helix; these metallointercalators may be usefully applied in probing DNA-mediated electron transfer. Here we describe a range of electron transfer reactions we carried out which are mediated by the DNA base paired stack. In some cases, DNA serves as a bridge, and spectroscopic analyses permit us to probe how the π stack couples DNA-bound donors and acceptors. These studies point to the sensitivity of coupling to DNA intercalation. However, if the DNA π stack effectively bridges donors and acceptors, the base-pair stack itself might serve not only as a conduit for electron transfer in DNA, but also in reactions initiated from a remote position. We carried out a series of reactions involving oxidative damage to DNA arising from the remotely positioned oxidant on the helix. The implications of long-range charge migration through DNA to effect damage are substantial. As in other DNA-mediated charge transfers, these reactions are highly dependent on DNA intercalation and the integrity of the intervening base-pair stack, but not on molecular distance. Furthermore, a physiologically important DNA lesion, the thymine dimers, can be reversed in a reaction initiated by electron transfer. This repair reaction can also be promoted from a distance as a result of long-range charge migration through the DNA base pair stack.