DNA损伤修复与细胞周期阻滞

面对各种因素导致的DNA损伤,细胞有一套应答修复机制。其中细胞周期阻滞在DNA损伤应答修复中扮演了重要角色,为修复受损DNA创造了条件。关于细胞周期调控的研究集中于细胞周期蛋白依赖性蛋白激酶(CDK)与细胞周期检查点等方面。在DNA损伤修复过程中,损伤位点募集的磷脂酰肌醇-3-激酶样激酶(PIKKs)可引起细胞周期检查点相关蛋白的激活,导致细胞周期阻滞。在碱基切除修复、核苷酸切除修复、错配修复、DNA双链断裂修复等常见的DNA损伤修复途径中,招募的损伤修复相关蛋白在细胞周期调控中也起到一定的作用。因此综述了主要DNA损伤修复形式与细胞周期阻滞之间的关系及研究进展。     

哺乳动物细胞DNA非同源末端连接及其生物学意义

DNA双链断裂是发生在哺乳动物细胞基因组水平上最严重的损伤。双链断裂得不到修复,细胞将会死亡或发生染色体断裂、丢失,若是错误修复将导致基因突变或基因组不稳定,增加癌症的风险度。哺乳动物细胞有两种重要的DNA双链断裂修复方式:非同源末端连接和同源重组。非同源末端连接途径除了在DNA双链断裂修复中起重要作用外,在V(D)J重组、HIV-1病毒整合宿主基因,以及假基因和重复序列的插入上也起着重要作用。由于非同源末端连接参与了机体许多重要的生理过程,因而其研究受到极大关注,并取得突破性的进展。     

应用彗星试验检测细胞DNA损伤的原理与方法

环境中多种因素，包括物理的如电离辐射和紫外线(UV)，化学的如多种氧化剂及三氯乙烷、丙烯酰氨等，以及混合性因素如老化、吸烟等均可直接或间接造成DNA链的断裂(single strand break，SSB)。虽然DNA单链断裂不会影响长链DNA分子的连续性，但双链断裂则会造成DNA片段的移位、异位、丢失等，从而引起遗传性的损伤并影响遗传行为。检测DNA链的断裂是测定DNA损伤程度的有效技术。     

人类DNA单链结合蛋白HSSB1与肿瘤关系的近期研究

细胞内DNA结构的完整性对于遗传物质的稳定、蛋白质的正确翻译以及细胞的生物学行为的维持至关重要.DNA损伤后的修复,特别是单链DNA结合蛋白(Humansingle-stranded DNA binding protein,HSSB1)在DNA损伤后的修复已经成为近年来国外学者研究的热点.缺乏HSSB1的细胞显示出DNA稳定性下降、对电离辐射(ionizing radiation,IR)敏感性增强、ATM检查点以下途径的活性丧失,紧随DNA损伤修复与DNA双链断裂同源重组(homologous recombination,HR)效率降低.研究还显示HSSB1结合并保护P21从泛素化介导的降解,与肝癌、肺癌和胃肠道癌症的发生存在负性关系.因此预测HSSB1可能用作恶性肿瘤治疗的靶点,并判定治疗效果的一种指标.鉴于此,本文就HSSB1在细胞中近期研究作一总结.     

DNA双链断裂修复与基因扩增关系的研究进展

基因扩增在肿瘤的发生发展过程中,起着不可忽视的作用.基因扩增的主要形式包括双微体(double minutes chromosomes,DMs)和均质染色区(homogeneous staining regions,HSRs).DNA双链断裂(DNA double strands break,DSBs)是最为严重的DNA损伤之一,近年来有关其与基因扩增关系的研究越来越多.该文综述了基因扩增的始动,并重点关注DNA双链断裂修复机制与基因扩增关系的研究进展.     

非同源末端连接研究进展及其在肿瘤发生和治疗中的意义

双链DNA损伤修复(DDR)途径主要包括非同源末端连接(NHEJ)和同源重组(HR),其中非同源末端连接(NHEJ)是DNA双链断裂后主要的修复方式,在原发肿瘤的发生发展和变异中发挥关键调控作用。近年发现了NHEJ修复DNA损伤新的组分和分子机制,丰富了对NHEJ通路的认识。通过对NHEJ分子机制及相关肿瘤的研究,发现抑制NHEJ活性可提高肿瘤细胞对放化疗的敏感性,提示NHEJ通路可能成为肿瘤治疗的新靶点。     

DNA构象多态性对肿瘤生物学行为影响的研究进展

获得性DNA损伤可导致体细胞基因突变或失活,与肿瘤的发生发展有关.传统的研究主要针对B-DNA双链结构中Watson-Crick碱基互补对的改变对DNA生物学的影响.而对DNA构象多态性(特殊DNA序列分子间或分子内氢键、不成对碱基配对等形成的多种非B-DNA三维立体空间结构)的研究尚少.对非B-DNA结构的生物学功能...     

DNA修复研究的回顾与前瞻

DNA是生命活动中最重要的遗传物质，也是环境因索攻击的靶分子，DNA损伤可归纳为四种主要类型；①碱基损伤，包括嘧啶二聚体、碱基共价结合物、减基烷基化和碱基脱落；②糖基破坏；③链断裂，包括DNA单键断裂、双链断裂必DNA使交联，包括DNA链内交联、链间交联、DNA蛋白质交联．DNA损伤直接影响复制、转录和蛋白质合成，进而影响细胞生长、发育、遗传、代谢和繁殖等生命活动，是造成突变、癌变、老化和死亡的重要原因．DNA修复是活细胞利用各种途径对其基因组出现的上述损伤的一种保护性的反应，在很大程度上保证了遗传物质基础的…     

用DNA多聚酶-1的Klenow片段原位检测脑缺血后神经元DNA的断裂揭示了神经元死亡的不同机制

程序性细胞死亡或凋亡是一个基本的生理学过程,其功能是在正常的发育和组织稳态过程中删除无价值或不能修复的损伤细胞。最近的研究表明:DNA链断裂可能与启动凋亡有关,这个断裂可能是单链(SSB)或是双链(DSB)。单链的损伤意味着可以修复;双链断裂是不能修复的结局,而且利用内源性Ca 依赖内切酶先于DNA裂解之前断裂,可以通过凝胶电泳检测。方法　36只雄性SD大鼠,在缺血再灌注的不同时间点随机分为6组。脑缺血后的DNA断裂用1Klenow标记法检测具有5′凸末端的单链DNA断裂或双链DNA断裂,2用TUNEL法检测具有3′凸末端或平端的DNA双…     

DNA损伤修复研究进展及其对围术期认知功能障碍的影响

DNA损伤与修复的动态平衡是保持基因组稳定性的重要因素之一.针对DNA双链断裂损伤(DSB),主要存在同源重组(HR)和非同源末端连接(NHEJ)两种修复方式.NHEJ是中枢神经系统成熟神经元DSB的主要修复途径.通过研究NHEJ分子机制和围术期认知功能障碍(POCD),发现围术期因素会影响NHEJ修复通路.NHEJ有...     

Genome instability and DNA damage accumulation in gene-targeted mice

Six major pathways for DNA repair have been identified. These include (1) DNA repair by direct reversal, (2) base excision repair, (3) mismatch repair, (4) nucleotide excision repair, (5) homologous recombination, and (6) non-homologous end-joining. In addition, several other cellular processes influence the response to DNA damage. The generation of gene-targeted organisms is crucial for assessing the relative contribution of single DNA repair proteins and DNA repair pathways in maintaining genome stability. In particular, the accumulation of DNA damage, mutations and cancer in unexposed gene-targeted animals illuminates the spontaneous load of a particular lesion and the relative significance of a single gene in a specific pathway. Strategies for the generation of gene-targeted mice have been available for 15 years and more than 100 different genes relevant to DNA repair have been targeted. This review describes some important progress made toward understanding spontaneous DNA damage and its repair, exemplified through one, or a few, gene-targeted mice from each major DNA repair pathway.     

Integrating the DNA damage and protein stress responses during cancer development and treatment

During evolution, cells have developed a wide spectrum of stress response modules to ensure homeostasis. The genome and proteome damage response pathways constitute the pillars of this interwoven ‘defensive’ network. Consequently, the deregulation of these pathways correlates with ageing and various pathophysiological states, including cancer. In the present review, we highlight: (1) the structure of the genome and proteome damage response pathways; (2) their functional crosstalk; and (3) the conditions under which they predispose to cancer. Within this context, we emphasize the role of oncogene‐induced DNA damage as a driving force that shapes the cellular landscape for the emergence of the various hallmarks of cancer. We also discuss potential means to exploit key cancer‐related alterations of the genome and proteome damage response pathways in order to develop novel efficient therapeutic modalities. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

An introduction for the special issue on environmental health and genome integrity

Environmental exposures and genome maintenance mechanisms that respond to environmentally-induced genotoxicity have a profound impact on human health. Eight review articles in this Special Issue (SI) titled “Environmental Health and Genome Integrity” describe emerging new mechanisms by which distinct forms of environmentally-induced DNA damage are remediated, and explain how DNA repair pathway choices impact genome integrity and disease propensity. Here, we provide an introduction to reviews from this SI. Our expanding knowledge of how genotoxic exposures impact the genome will allow us to better predict, prevent and treat environmentally-induced human diseases such as cancer and neurodegenerative disorders.     

Fanconi anemia and the underlying causes of genomic instability

Fanconi anemia (FA) is a rare genetic disorder, characterized by birth defects, progressive bone marrow failure, and a predisposition to cancer. This devastating disease is caused by germline mutations in any one of the 22 known FA genes, where the gene products are primarily responsible for the resolution of DNA interstrand cross-links (ICLs), a type of DNA damage generally formed by cytotoxic chemotherapeutic agents. However, the identity of endogenous mutagens that generate DNA ICLs remains largely elusive. In addition, whether DNA ICLs are indeed the primary cause behind FA phenotypes is still a matter of debate. Recent genetic studies suggest that naturally occurring reactive aldehydes are a primary source of DNA damage in hematopoietic stem cells, implicating that they could play a role in genome instability and FA. Emerging lines of evidence indicate that the FA pathway constitutes a general surveillance mechanism for the genome by protecting against a variety of DNA replication stresses. Therefore, understanding the DNA repair signaling that is regulated by the FA pathway, and the types of DNA lesions underlying the FA pathophysiology is crucial for the treatment of FA and FA-associated cancers. Here, we review recent advances in our understanding of the relationship between reactive aldehydes, bone marrow dysfunction, and FA biology in the context of signaling pathways triggered during FA-mediated DNA repair and maintenance of the genomic integrity. Environ. Mol. Mutagen. 2020. © 2020 Wiley Periodicals, Inc.     

DNA break-induced sumoylation is enabled by collaboration between a SUMO ligase and the ssDNA-binding complex RPA

Upon genome damage, large-scale protein sumoylation occurs from yeast to humans to promote DNA repair. Currently, the underlying mechanism is largely unknown. Here we show that, upon DNA break induction, the budding yeast SUMO ligase Siz2 collaborates with the ssDNA-binding complex RPA (replication protein A) to induce the sumoylation of recombination factors and confer damage resistance. Both RPA and nuclease-generated ssDNA promote Siz2-mediated sumoylation. Mechanistically, the conserved Siz2 interaction with RPA enables Siz2 localization to damage sites. These findings provide a molecular basis for recruiting SUMO ligases to the vicinity of their substrates to induce sumoylation upon DNA damage.     

Super‐resolution imaging reveals changes in Escherichia coli SSB localization in response to DNA damage

The E. coli single‐stranded DNA‐binding protein (SSB) is essential to viability. It plays key roles in DNA metabolism where it binds to nascent single strands of DNA and to target proteins known as the SSB interactome. There are >2,000 tetramers of SSB per cell with 100–150 associated with the genome at any one time, either at DNA replication forks or at sites of repair. The remaining 1,900 tetramers could constantly diffuse throughout the cytosol or be associated with the inner membrane as observed for other DNA metabolic enzymes. To visualize SSB localization and to ascertain potential spatiotemporal changes in response to DNA damage, SSB‐GFP chimeras were visualized using a novel, super‐resolution microscope optimized for the study of prokaryotic cells. In the absence of DNA damage, SSB localizes to a small number of foci and the excess protein is associated with the inner membrane where it binds to the major phospholipids. Within five minutes following DNA damage, the vast majority of SSB disengages from the membrane and is found almost exclusively in the cell interior. Here, it is observed in a large number of foci, in discreet structures or, in diffuse form spread over the genome, thereby enabling repair events.     

ATR phosphorylates SMARCAL1 to prevent replication fork collapse

The DNA damage response kinase ataxia telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of cancer cells, making ATR a potential therapeutic target. Here we use selective ATR inhibitors to demonstrate that acute inhibition of ATR kinase activity yields rapid cell lethality, disrupts the timing of replication initiation, slows replication elongation, and induces fork collapse. We define the mechanism of this fork collapse, which includes SLX4-dependent cleavage yielding double-strand breaks and CtIP-dependent resection generating excess single-stranded template and nascent DNA strands. Our data suggest that the DNA substrates of these nucleases are generated at least in part by the SMARCAL1 DNA translocase. Properly regulated SMARCAL1 promotes stalled fork repair and restart; however, unregulated SMARCAL1 contributes to fork collapse when ATR is inactivated in both mammalian and Xenopus systems. ATR phosphorylates SMARCAL1 on S652, thereby limiting its fork regression activities and preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR prevents fork collapse, promotes the completion of DNA replication, and maintains genome integrity.     

The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks

Mutations in SMARCAL1 (HARP) cause Schimke immunoosseous dysplasia (SIOD). The mechanistic basis for this disease is unknown. Using functional genomic screens, we identified SMARCAL1 as a genome maintenance protein. Silencing and overexpression of SMARCAL1 leads to activation of the DNA damage response during S phase in the absence of any genotoxic agent. SMARCAL1 contains a Replication protein A (RPA)-binding motif similar to that found in the replication stress response protein TIPIN (Timeless-Interacting Protein), which is both necessary and sufficient to target SMARCAL1 to stalled replication forks. RPA binding is critical for the cellular function of SMARCAL1; however, it is not necessary for the annealing helicase activity of SMARCAL1 in vitro. An SIOD-associated SMARCAL1 mutant fails to prevent replication-associated DNA damage from accumulating in cells in which endogenous SMARCAL1 is silenced. Ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) phosphorylate SMARCAL1 in response to replication stress. Loss of SMARCAL1 activity causes increased RPA loading onto chromatin and persistent RPA phosphorylation after a transient exposure to replication stress. Furthermore, SMARCAL1-deficient cells are hypersensitive to replication stress agents. Thus, SMARCAL1 is a replication stress response protein, and the pleiotropic phenotypes of SIOD are at least partly due to defects in genome maintenance during DNA replication.     

USP51 deubiquitylates H2AK13,15ub and regulates DNA damage response

Dynamic regulation of RNF168-mediated ubiquitylation of histone H2A Lys13,15 (H2AK13,15ub) at DNA double-strand breaks (DSBs) is crucial for preventing aberrant DNA repair and maintaining genome stability. However, it remains unclear which deubiquitylating enzyme (DUB) removes H2AK13,15ub. Here we show that USP51, a previously uncharacterized DUB, deubiquitylates H2AK13,15ub and regulates DNA damage response. USP51 depletion results in increased spontaneous DNA damage foci and elevated levels of H2AK15ub and impairs DNA damage response. USP51 overexpression suppresses the formation of ionizing radiation-induced 53BP1 and BRCA1 but not RNF168 foci, suggesting that USP51 functions downstream from RNF168 in DNA damage response. In vitro, USP51 binds to H2A–H2B directly and deubiquitylates H2AK13,15ub. In cells, USP51 is recruited to chromatin after DNA damage and regulates the dynamic assembly/disassembly of 53BP1 and BRCA1 foci. These results show that USP51 is the DUB for H2AK13,15ub and regulates DNA damage response.     

Electromagnetic fields and DNA damage

A major concern of the adverse effects of exposure to non-ionizing electromagnetic field (EMF) is cancer induction. Since the majority of cancers are initiated by damage to a cell's genome, studies have been carried out to investigate the effects of electromagnetic fields on DNA and chromosomal structure. Additionally, DNA damage can lead to changes in cellular functions and cell death. Single cell gel electrophoresis, also known as the ‘comet assay’, has been widely used in EMF research to determine DNA damage, reflected as single-strand breaks, double-strand breaks, and crosslinks. Studies have also been carried out to investigate chromosomal conformational changes and micronucleus formation in cells after exposure to EMF. This review describes the comet assay and its utility to qualitatively and quantitatively assess DNA damage, reviews studies that have investigated DNA strand breaks and other changes in DNA structure, and then discusses important lessons learned from our work in this area.