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。     

TopBP1 and DNA polymerase-α directly recruit the 9-1-1 complex to stalled DNA replication forks

TopBP1 and the Rad9–Rad1–Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR–ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-α to sites of replication stress. Furthermore, we show that pol-α is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-α, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.     

DNA与个体发育调控(1)

多细胞生物的身体都是由一个细胞发育而来的,因此该细胞内的遗传物质DNA应携带有建造身体结构的全部信息指令。然而,在DNA序列中却只有为基因编码的序列,以及控制基因表达时间、地点、多少的调控序列,并没有如何建造身体的具体信息指令。DNA的蓝图作用,是通过各种蛋白质分子的顺序表达来形成各种类型的细胞,同时通过一些细胞分泌的信息分子指挥周围细胞表达出能够产生机械力的蛋白质分子,让细胞按照特异方式彼此结合,从而实现个体发育的精准调控。     

光生物素标记DNA探针检测感染细胞的BHV—1 DNA

将七株从不同地区、不同牛种或不同部位分离出的牛Ⅰ型疱疹病毒(BHV-1)培养于MDBK细胞中,从感染的细胞中提取病毒并抽提其DNA将克隆的BHV-1LA株DNA的HindⅢ—Ⅰ片段(11.7Kb)用光生物素标记作为探针,检测上述七株感染细胞的BHV-1 DNA,其中有六株(均属IBR临床型)呈阳性反应,另一株(属IPP临床型)呈阴性反应,这一探针将作为一种有效的检疫工具在本病的防制中起重要作用.     

甲型H1N1流感病毒DNA序列碱基的预测

甲型流感H1N1亚型曾给人类带来了重大灾难。本文提出了一种利用时间序列模型预测碱基的方法,对所选取的1970年～2010年同源性相对较高的41条H1N1流感病毒数据利用ARIMA(p,d,q)模型对前20个位置去拟合并且预测,除极个别外由预报区域图显示原始数据都在预报区域内,表明模型建立的比较合理,预报效果很好,这对H1N1病毒的研究有着重要的意义。     

XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.     

WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response

WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH^−/−) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.     

BRCA1与DNA损伤修复调控网络

DNA损伤应答机制的存在有助于机体基因组稳定性的维持. BRCA1是一种重要的肿瘤抑制基因,它在DNA损伤应答中发挥了重要的作用. BRCA1可以与BARD1结合形成稳定的异源二聚体,作为BRCA1复合体蛋白组分的核心参与了DNA损伤信号传递、同源重组修复、DNA复制、细胞周期等多途径的调控.本文主要对BRCA1功能及其参与DNA损伤应答网络调控展开阐述,并总结了利用PARP抑制剂针对BRCA1突变肿瘤进行治疗产生耐药性的多种机制.     

实验1 哺乳类高分子量DNA的提取

分子遗传学和基因工程已日益成为国内遗传学教学和科研的重要组成部分,为了有效地提高教学质量和科研水平,除了讲授这些领域中的基本原理和最新进展外,很重要的是应该广泛开设有关的实验,推广和交流实验操作技术,以便让更多的遗传学工作者掌握和运用分子遗传学和基因工程的技术去开展工作。为此,我们应《遗传》编辑部之约,准备分别介绍哺乳类基因工程的十几个基本实验操作,分期刊登在《遗传》上。但是,基因工程所用技术涉及的学科领域很多,即使是同一项实验,不同实验室的操作程序也不尽相同;再加上这方面的进展堪称是日新月异,不断地有改进和创新,因此,这里介绍的是其中的一部分,而且只是我们实验室所熟悉惯用的操作。沧海遗珠,在所难免,挂一漏万,祈请鉴宥。凡有错误不妥之处,还请批评指正。     

Interaction between DNA Polymerase β and BRCA1

The breast cancer 1 (BRCA1) protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB) repair pathways and have recently included base excision repair (BER). However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS) treatment of cells and the BER enzyme DNA polymerase β (pol β). MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol β in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol β were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol β and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol β was similar in BRCA1 positive and negative cells. However, a fraction of pol β initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol β expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair.     

BRCA1蛋白参与DNA双链损伤应答

BRCA1是乳腺癌易感基因,负责维持细胞基因组的稳定性,防止调控细胞增殖和肿瘤生长的基因突变的积累。BRCA1基因蛋白产物结构复杂,功能多样,是细胞内重要的多功能蛋白,参与执行多种生理代谢过程。本主要探讨了BRCA1蛋白应答DNA双链损伤过程中所伴随的一系列信号传导的历程,阐述了连续的生理生化反应中BRCA1蛋白所发挥的作用。     

利用PCR法检测HIV—1DNA

通过比较多个HIV(人免疫缺陷病毒)分离株的核苷酸序列,我们选择膜基因上7373—7514位的一段保守区为目的片段合成了引物1(5′—AGCAGCAGGAAGCACTATGGGC—3′)和引物2(5′—CCAGACTGTGAGTTGCAACA—3′),并分别以质粒PⅢexE7和MT4-HIV-1 DNA,为模板进行了PCR反应及敏感性试验。结果表明,用PCR法可检测出1～10个质粒分子及1×10~6个细胞中一个感染细胞。因此我们推论,本法可应用于AIDS临床标本的检测。     

GADD45α inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair

In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45α. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45α, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45α during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45α increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45α specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45α. Since GADD45α binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair.     

DNA修复通路的关键酶——FAN1

<正>来自德克萨斯大学M.D.安德森癌症中心和浙江大学生命科学院的科学家报道发现了一种可修复DNA交联损伤的关键酶.这一成果将刊登在Science上."DNA交联损伤的修复在肿瘤中十分常见,比如乳腺     

53BP1与DNA双链断裂损伤修复

DNA的精确复制和遗传对维持基因组稳定性有重要作用。DNA双链断裂损伤可能诱导细胞凋亡和染色质重排,在肿瘤的发生发展过程中发挥作用。53BP1是DNA双链断裂修复中的重要调节蛋白质之一,对调控损伤修复平衡和维持基因组稳定性起着重要作用。本文主要对53BP1的结构、生物学功能、信号通路、分子机制和翻译后修饰做一浅显的总结和展望,希望能为53BP1的深入研究提供一些理论基础。     

PHF1在肿瘤DNA损伤修复中的作用

轻微的DNA损伤可启动损伤修复途径,严重的DNA损伤则会启动细胞休眠或凋亡途径。PHF1是PcG蛋白家族中的重要组分,参与复杂的生物学过程,包括DNA损伤修复、细胞休眠或凋亡、组蛋白翻译后修饰和染色体重排。本文主要对PHF1的结构、参与的信号通路、翻译后修饰及生物学功能做小结和展望,为PHF1进一步研究提供理论基础。     

Poly(ADP-ribose) Polymerase-1 Inhibits Strand-Displacement Synthesis of DNA Catalyzed by DNA Polymerase β

Poly(ADP-ribose) polymerase-1 (PARP-1), a eucaryotic nuclear DNA-binding protein that is activated by breaks in DNA chains, may be involved in the base excision repair (BER) because DNAs containing single-stranded gaps and breaks are intermediates of BER. The effect of PARP-1 on the DNA synthesis catalyzed in vitro by DNA polymerase beta (pol beta) was studied using analogs of DNA substrates produced during BER and imitating intermediates of the short patch and long patch subpathways of BER. Oligonucleotide duplexes of 34 bp that contained a mononucleotide gap or a single-strand break with tetrahydrofuran phosphate or phosphate at the 5;-end of the downstream oligonucleotide were taken as DNA substrates. The efficiency of DNA synthesis was determined at various ratios of pol beta and PARP-1. The efficiency of gap filling was decreased in the presence of PARP-1, but strand-displacement DNA synthesis was inhibited significantly stronger, which seemed to be due to competition between PARP-1 and pol beta for DNA. In the presence of NAD+ and single-strand breaks in DNA, PARP-1 catalyzes the synthesis of poly(ADP-ribose) covalently attached to the enzyme, and this automodification is thought to provide for dissociation of PARP-1 from DNA. The effect of PARP-1 automodification on inhibition of DNA synthesis was studied, and efficiency of mononucleotide gap filling was shown to be restored, but strand-displacement synthesis did not revert to the level observed in the absence of PARP-1. PARP-1 is suggested to regulate the interaction between pol beta and DNA, in particular, via its own automodification.