DNA错配修复

ＤＮＡ错配修复＊任庆虎张宗玉童坦君（北京医科大学生化与分子生物学系,北京１０００８３）关键词错配修复遗传性非息肉型结直肠癌微卫星ＤＮＡＤＮＡ错配修复基因（ＤＮＡｍｉｓｍａｔｃｈｒｅｐａｉｒｇｅｎｅ）首先在细菌和酵母中发现,最近在人类基因组中也找到了类...     

DNA错配修复与肺癌

肺癌是目前世界上最常见的恶性肿瘤之一,虽然近年来对其研究较多,但其发生发展的确切机制仍不清楚。DNA错配修复作为一种重要的复制后修复系统,在确保DNA复制保真性、控制基因突变和维持基因组稳定等方面具有重要作用。近年研究表明,DNA错配修复系统与肺癌的发生、治疗及预后判断有着密切关系。本文主要对DNA错配修复系统在肺癌中的研究进展作一简要综述。     

一组多功能的错配修复蛋白

错配修复蛋白是DNA错配修复系统中主要功能蛋白质,主要参与DNA复制过程中对错配碱基的识别和修复.近年来研究表明错配修复蛋白还参与DNA损伤信号的传递、细胞周期的调控、减数分裂和有丝分裂等.错配修复蛋白缺陷会增加患肿瘤的危险性或者直接导致肿瘤;由于错配修复蛋白参与了DNA损伤信号传递、周期调控,错配修复蛋白缺陷还会导致细胞对相关抗癌药物产生耐受.     

DNA错配修复基因mutS的高效表达及表达产物活性鉴定

将DNA错配修复基因mutS(2.56kb)克隆于分泌型原核表达载体pET32a( )上,以N端融合6个组氨酸的形式在E.col AD494(DE3)中进行了IPTG诱导表达。SDS－PAGE分析证实有一与预期分子量相应的诱导表达条带,其表达量占全菌蛋白质的35％左右,且表达蛋白以可溶形式存在。利用固定化金属离子（Ni^2 )配体亲和层析柱纯化目的蛋白,其纯度为90％以上。与含有错配碱基DNA双链的结合反应证明该蛋白具有特异性识别,结合含有错配碱基DNA双链的生物活性。     

p53与癌症治疗

p53蛋白是一种重要的肿瘤抑制蛋白,它可以诱导肿瘤细胞生长停滞、衰老以及程序化死亡.由于它在癌细胞抑制中的至关重要作用,因而目前关于癌症的治疗大多数都是直接或间接通过调控p53蛋白来实现的.文中主要介绍了近几年来p53蛋白在理论和临床上最新的研究进展.     

利用细胞凋亡原理治疗癌症

细胞凋亡(apoptosis)是致细胞死亡的重要机制之一,是细胞一种生理性、主动性的“自觉自杀行为”,犹如秋天片片树叶的“凋落”。这些细胞死得有规律,似乎是按编好了的“程序”进行的,所以又称之为“程序性细胞死亡”。它受细胞内部基因调控,而调控凋亡的基因有两类：(1)抑制细胞基因凋亡;(2)启动或促进细胞凋亡。基于此,可利用调控细胞凋亡基因的后以启动和促进细胞凋亡。     

激发p53对癌症开战

有时被称为“基因组守护神”的肿瘤抑制蛋白p53,针对DNA损伤发生反应,要么停止细胞分裂,要么促使细胞凋亡。p53的反应通过阻止已发生恶性突变的细胞不停的生长,从而阻断肿瘤的结构形成。但是p53自身也对损伤敏感,这一点被认为利于半数癌症的生长,包括常见的一些癌症,如皮肤癌、乳腺癌和结肠癌。现在,研究人员已鉴定了一种药物,能够保留一些变异的P53蛋白的正常功能,因此可能开辟出一条新的治疗癌症的方法。     

DNA错配修复系统研究进展

DNA错配修复(mismatch repair, MMR)系统广泛存在于生物体中.从原核生物大肠杆菌到真核生物及人类,MMR系统有不同的组成成分和修复机制.人体内MMR基因缺陷会造成基因组的不稳定并诱发遗传性非息肉型直肠癌以及其他自发性肿瘤.大肠杆菌MMR系统中的MutS蛋白可特异识别错配或未配对碱基,目前已经发展了多种基于MutS蛋白的基因突变/多态性检测技术.     

癌症DNA甲基化调控位点的识别

DNA甲基化是一种重要的表观遗传学修饰,在基因的转录调控方面具有重要的作用。异常的DNA甲基化可以导致癌症等复杂疾病发生,癌基因相关的DNA甲基化调控位点的识别对于解析癌症的发生发展机制及识别新的癌症标记具有重要意义。本研究通过整合The Cancer Genome Atlas(TCGA)的泛癌症基因组的高通量甲基化谱和基因表达谱,识别癌基因相关的DNA甲基化调控位点。对于每种癌症分批次计算Cp G位点甲基化与相关基因表达之间的相关性,并筛选调控下游基因的Cp G位点(包括强调控位点、弱调控位点和不调控位点),结果表明仅有一半的Cp G位点对下游基因具有调控作用;对癌症间共享的调控位点的分析发现不同癌症间共享的调控位点不尽相同,表明癌症特异的甲基化调控位点的存在。进一步地,对差异甲基化和差异表达基因的功能富集分析揭示了受甲基化调控的基因确实参与了癌症发生发展相关的功能。本研究的结果是对当前甲基化调控位点集的重要补充,也是识别癌症新型分子标记特征的重要资源。     

Cisplatin resistance: Preclinical findings and clinical implications

Cisplatin is used for the treatment of many types of solid cancers. While testicular cancers respond remarkably well to cisplatin, the therapeutic efficacy of cisplatin for other solid cancers is limited because of intrinsic or acquired drug resistance. Our understanding about the mechanisms underlying cisplatin resistance has largely arisen from studies carried out with cancer cell lines in vitro. The process of cisplatin resistance appears to be multifactorial and includes changes in drug transport leading to decreased drug accumulation, increased drug detoxification, changes in DNA repair and damage bypass and/or alterations in the apoptotic cell death pathways. Translation of these preclinical findings to the clinic is emerging, but still scarce. The present review describes and discusses the clinical relevance of in vitro models by comparing the preclinical findings to data obtained in clinical studies.     

Cadmium inhibits human DNA mismatch repair in vivo

The heavy metal cadmium (Cd) is a human carcinogen that inhibits DNA repair activities. We show that DNA mismatch repair (MMR)-mediated cell cycle arrest after alkylation damage is suppressed by exposure to Cd and that this effect is reversed by preincubation with excess of zinc (Zn). We show that Cd-mediated inactivation of MMR activity is not caused by disruption of complex formation between the MMR proteins hEXO1-hMutS alpha and hEXO1-hMutL alpha nor does Cd inhibit 5'-exonuclease activity of hEXO1 in vitro. Thus, our studies show that exposure of human cells to Cd suppresses MMR activity, a repair activity known to play an important role in colon cancer and that this effect can be reversed by Zn treatment.     

A New Isoquinolinium Derivative, Cadein1, Preferentially Induces Apoptosis in p53-defective Cancer Cells with Functional Mismatch Repair via a p38-dependent Pathway

We screened a protoberberine backbone derivative library for compounds with anti-proliferative effects on p53-defective cancer cells. A compound identified from this small molecule library, cadein1 (cancer-selective death inducer 1), an isoquinolinium derivative, effectively leads to a G₂/M delay and caspase-dependent apoptosis in various carcinoma cells with non- functional p53. The ability of cadein1 to induce apoptosis in p53-defective colon cancer cells was tightly linked to the presence of a functional DNA mismatch repair (MMR) system, which is an important determinant in chemosensitivity. Cadein1 was very effective in MMR⁺/p53⁻ cells, whereas it was not effective in p53⁺ cells regardless of the MMR status. Consistently, when the function of MMR was blocked with short hairpin RNA in SW620 (MMR⁺/p53⁻) cells, cadein1 was no longer effective in inducing apoptosis. Besides, the inhibition of p53 increased the pro-apoptotic effect of cadein1 in HEK293 (MMR⁺/p53⁺) cells, whereas it did not affect the response to cadein1 in RKO (MMR⁻/p53⁺) cells. The apoptotic effects of cadein1 depended on the activation of p38 but not on the activation of Chk2 or other stress-activated kinases in p53-defective cells. Taken together, our results show that cadein1 may have a potential to be an anti-cancer chemotherapeutic agent that is preferentially effective on p53-mutant colon cancer cells with functional MMR.     

DNA repair protein O6-methylguanine-DNA methyltransferase in testis and testicular tumors as determined by a novel nonradioactive assay

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT, alkyltransferase) is an important suicide enzyme involved in defense against O6-alkylating endogenous metabolites and environmental carcinogens. It also plays a pivotal role in primary and acquired resistance of tumors to alkylating anticancer drugs targeting the O6-position of guanine (i.e., methylating and chloroethylating agents). MGMT can thus be considered a crucial biomarker for individual susceptibility to alkylating carcinogens and tumor drug resistance. This implies a need for a fast and convenient method for determination of MGMT. Routinely, MGMT is being quantified by radioactive assays which are relatively laborious. Here we report a nonradioactive MGMT enzyme-linked immunosorbent assay (ELISA) for quantification of MGMT in cell and tissue homogenates. We compared the MGMT-ELISA with the standard radioactive assay and found it to be as sensitive but less time consuming. Therefore, it represents an alternative for the quantification of MGMT in cell and tissue homogenates. We applied the assay for determining MGMT in normal and tumor tissue of testes. In both normal and tumor tissue MGMT was quite variable, ranging from zero to 1300 fmol/mg protein. In various tumor samples MGMT was lower than MGMT in the normal tissue from the same patient or was even not detectable. The MGMT-ELISA might become a useful tool for MGMT determination in clinical routine and health control.     

Formation of a DNA mismatch repair complex mediated by ATP

The mismatch repair proteins, MutS and MutL, interact in a DNA mismatch and ATP-dependent manner to activate downstream events in repair. Here, we assess the role of ATP binding and hydrolysis in mismatch recognition by MutS and the formation of a ternary complex involving MutS and MutL bound to a mismatched DNA. We show that ATP reduces the affinity of MutS for mismatched DNA and that the modulation of DNA binding affinity by nucleotide is even more pronounced for MutS E694A, a protein that binds ATP but is defective for ATP hydrolysis. Despite the ATP hydrolysis defect, E694A, like WT MutS, undergoes rapid, ATP-dependent dissociation from a DNA mismatch. Furthermore, MutS E694A retains the ability to interact with MutL on mismatched DNA. The recruitment of MutL to a mismatched DNA by MutS is also observed for two mutant MutL proteins, E29A, defective for ATP hydrolysis, and R266A, defective for DNA binding. These results suggest that ATP binding in the absence of hydrolysis is sufficient to trigger formation of a MutS sliding clamp. However, recruitment of MutL results in the formation of a dynamic ternary complex that we propose is the intermediate that signals subsequent repair steps requiring ATP hydrolysis.     

Analysis of in vivo correction of defined mismatches in the DNA mismatch repair mutants msh2 , msh3 and msh6 of Saccharomyces cerevisiae

We have analysed the correction of defined mismatches in wild-type and msh2, msh3, msh6 and msh3 msh6 mutants of Saccharomyces cerevisiae in two different yeast strain backgrounds by transformation with plasmid heteroduplex DNA constructs. Ten different base/base mismatches, two single-nucleotide loops and a 38-nucleotide loop were tested. Repair of all types of mismatches was severely impaired in msh2 and msh3 msh6 mutants. In msh6 mutants, repair efficiency of most base/base mismatches was reduced to a similar extent as in msh3 msh6 double mutants. G/T and A/C mismatches, however, displayed residual repair in msh6 mutants in one strain background, implying a role for Msh3p in recognition of base/base mismatches. Furthermore, the efficiency of repair of base/base mismatches was considerably reduced in msh3 mutants in one strain background, indicating a requirement for MSH3 for fully efficient mismatch correction. Also the efficiency of repair of the 38-nucleotide loop was reduced in msh3 mutants, and to a lesser extent in msh6 mutants. The single-nucleotide loop with an unpaired A was less efficiently repaired in msh3 mutants and that with an unpaired T was less efficiently corrected in msh6 mutants, indicating non-redundant functions for the two proteins in the recognition of single-nucleotide loops. Received: 7 August 1997 / Accepted: 24 September 1997     

从DNA修复机理看细胞癌变的发生机制

DNA损伤是引起基因突变,导致细胞恶性转化的重要原因．DNA损伤的修复过程非常复杂,是与细胞周期调节、DNA复制和DNA转录等生命活动紧密相连的．首先DNA修复需要细胞周期停滞,避免DNA损伤进入子代细胞．其次,参与DNA转录的某些基因产物参与DNA损伤的识别,有利于转录链的优先修复．最后,DNA修复系统NER、MMR参与损伤修复．上述DNA修复过程任何环节的异常,都将造成DNA修复功能减弱,导致某些功能基因突变,从而导致细胞的恶性转化．