错配修复蛋白在DNA双链断裂修复中的作用

DNA双链断裂(DSBs)是最严重的DNA损伤之一,近年来受到人们广泛的关注.错配修复(MMR)系统广泛存在于生物体中,是细胞复制后的一种修复方式,通过矫正在DNA复制和重组过程中产生的碱基对错配和小的核苷酸插入或缺失而保持基因组的稳定性.研究发现MMR系统在DSBs修复中起着重要的作用,MMR蛋白通过与同源重组(HR)和非同源末端连接(NHEJ)修复相互作用参与DSBs修复.本文重点关注MMR通路几种关键蛋白在DSBs修复中的作用.     

胃癌中hMLH1、hMSH2、PTEN和PCNA表达的相关性及意义

近年来,研究发现除了一些癌基因基因以外,错配修复(m ism atch repair,MMR)基因也与胃癌的发生与演进密切相关。研究证实,MMR基因能够纠正DNA复制过程中的碱基错配,避免癌相关基因的突变,保护基因的完整性与稳定性,从而抑制癌的发生。在已经发现的MMR基因中,hMLH1和hMSH2与胃癌     

DNA错配修复与肿瘤的发生及治疗

DNA错配修复系统能够识别和纠正错配的DNA碱基对,确保DNA复制过程的保真性.若是错配修复系统存在缺陷将导致基因突变或基因组不稳定性,最终会导致肿瘤的产生.错配修复系统不仅可以通过修复在DNA复制和重组过程中产生的碱基错配来维持基因组的稳定性,还可以通过识别DNA损伤介导细胞的凋亡,所以细胞的错配修复功能与化疗疗效也有密切相关性.错配修复系统对肿瘤诊断、治疗及预后的重要价值决定了它在肿瘤研究中的重要性.     

DNA错配修复蛋白多功能性的研究进展

DNA错配修复(mismatch repair,MMR)系统是DNA损伤修复的多种途径之一,存在于从细菌、酵母到人体的所有生物体,由一组高保守性酶蛋白组成.其通过校正DNA复制及重组中产生的碱基错配与插入/缺失环,维持所有生物基因组稳定性的功能已研究比较清楚.越来越多的研究还揭示了错配修复蛋白的其他功能:参与调控DNA损伤应答,同源重组,减数分裂的染色体配对和分离,抗体多样性产生及三核苷酸重复序列扩增等过程.本文将对错配修复蛋白多功能性的研究进展作一综述.     

人错配修复基因hMLH1研究的新进展

人错配修复基因(mismatch repair,MMR)的主要功能是对DNA链中因某些原因造成的错误配对进行修复.目前已知MMR主要有hMLH1、hMSH2、hMSH6、hPMS2等.它们能够识别和修复在DNA复制过程中因插入、缺失或单核苷酸突变形成的错配,从而大大减低基因组微卫星不稳定性(MSI),维持基因组的稳定性.     

DNA复制错误与肿瘤

对ＤＮＡ复制过程中形成的碱基错配进行修复是生物界普遍存在的一种保持遗传忠实性、调节遗传多样性的基本功能。ＤＮＡ错配修复（ｍｉｓｍａｔｃｈｒｅｐａｉｒ，ＭＭＲ）基因是细胞内负责对碱基错配进行修复的基因，它们的缺陷可导致细胞突变频率的增加而形成突变子表型...     

PIKK调控DNA双链断裂修复的研究进展

生物体细胞基因组完整性受到诸多因素的威胁,包括DNA复制过程中DNA碱基错配、化学物质产生的碱基加合物(adduct formation)和交叉链(cross-links)、紫外线诱导的碱基损伤、电离辐射导致的DNA单链或双链断裂等。DNA双链断裂(DNAdouble-strand break,DSB)被认为是细胞毒性最强的DNA损伤。     

DNA错配修复基因MSH3和MSH6在肠道肿瘤发生中共同起着抑制作用

人类遗传性非息肉型结肠癌 ( HNPCC)是常染色体显性遗传疾病 ,HNPCC病人的肿瘤存在着DNA错配修复缺陷。近年来对错配修复基因MSH2、MLH1、PMS2研究的比较多 ,但对 MSH6和 MSH3的研究相对比较少。已有文章表明MSH6突变的小白鼠有肿瘤易感的表型 ,为研究MSH3的作用 ,作者设计了 MSH6和 MSH3突变的小白鼠 ,并核查它们的表现型。实验中作者发现 MSH3- /-的小白鼠的细胞提取物有能力修复单个核苷酸错配和单个核苷酸插入与缺失错配 ,但不能修复更大段的插入和缺失错配。而 MSH6- /-细胞提取物不能修复碱基错配 ,但能有效地修复…     

基因组不稳定性与肿瘤

基因组结构的相对稳定是生物种系得以维持和延续的基本前提和重要保证，细胞内逐渐形成了一整套有效的机制以保证遗传信息稳定而真实地代代相传。对DNA复制过程中形成的碱基错配进行修复是生物界普遍存在的一种保持遗传忠实性、调节遗传多样性的基本功能。错配修复基因是细胞内负责对减基错配进行修复的基因，它们的缺陷可导致细胞突变频率的增加而形成突变子表型，最为常见的突变子表型为微随体DNA的复制错误，而后者可导致基因组不稳定性．最近．通过对遗传性非息肉性结直肠癌（HNPCC）的分子发病机理的研究，人们已克隆出与HNPCC发病有关的错配修复基因hMSH2、hMLH1、hPMS1和hPMS2、，它们的缺陷分别在50～60％、30％、5％和5％的HNNPCC中起作用。通过对错配修复基因的研究，人们有可能确定某些肿瘤发病的危险性，从而采取相应的措施以达到降低死亡率、提高生存率的目的。     

错配修复系统的生物学效应和机理

在ＤＮＡ正常代谢中，碱基错配、插入或缺失可导致基因组ＤＮＡ复制错误。已证明在细菌、酵母及高等真核细胞中，都存在错配修复系统。这方面的研究已取得了一些进展，包括Ｅ．ｃｏｌｉ．中的Ｍｕｔ ＨＬＳ修复系统，酵母中的错配修主其蛋白以及真核细胞中的错配修复基因及蛋白。这对消除ＤＮＡ生物合成错误，增加染色体复制的可信性，防止自由突出以及肿瘤的发生发展、诊断和治疗有着重要意义。     

Mismatch repair in Gram-positive bacteria

DNA mismatch repair (MMR) is responsible for correcting errors formed during DNA replication. DNA polymerase errors include base mismatches and extra helical nucleotides referred to as insertion and deletion loops. In bacteria, MMR increases the fidelity of the chromosomal DNA replication pathway approximately 100-fold. MMR defects in bacteria reduce replication fidelity and have the potential to affect fitness. In mammals, MMR defects are characterized by an increase in mutation rate and by microsatellite instability. In this review, we discuss current advances in understanding how MMR functions in bacteria lacking the MutH and Dam methylase-dependent MMR pathway.     

DNA mismatch repair: molecular mechanism, cancer, and ageing

DNA mismatch repair (MMR) proteins are ubiquitous players in a diverse array of important cellular functions. In its role in post-replication repair, MMR safeguards the genome correcting base mispairs arising as a result of replication errors. Loss of MMR results in greatly increased rates of spontaneous mutation in organisms ranging from bacteria to humans. Mutations in MMR genes cause hereditary nonpolyposis colorectal cancer, and loss of MMR is associated with a significant fraction of sporadic cancers. Given its prominence in mutation avoidance and its ability to target a range of DNA lesions, MMR has been under investigation in studies of ageing mechanisms. This review summarizes what is known about the molecular details of the MMR pathway and the role of MMR proteins in cancer susceptibility and ageing.     

DNA repair in antibody somatic hypermutation

Somatic hypermutation (SHM) underlies the generation of a diverse repertoire of high-affinity antibodies. It is effected by a two-step process: (i) DNA lesions initiated by activation-induced cytidine deaminase (AID), and (ii) lesion repair by the combined intervention of DNA replication and repair factors that include mismatch repair (MMR) proteins and translesion DNA synthesis (TLS) polymerases. AID and TLS polymerases that are crucial to SHM, namely polymerase (pol) theta, pol zeta and pol eta, are induced in B cells by the stimuli that are required to trigger this process: B-cell receptor crosslinking and CD40 engagement by CD154. These polymerases, together with MMR proteins and other DNA replication and repair factors, could assemble to form a multimolecular complex ("mutasome") at the site of DNA lesions. Molecular interactions in the mutasome would result in a "polymerase switch", that is, the substitution of the high-fidelity replicative pol delta and pol epsilon with the TLS pol theta, pol eta, Rev1, pol zeta and, perhaps, pol iota, which are error-prone and crucially insert mismatches or mutations while repairing DNA lesions. Here, we place these concepts in the context of the existing in vivo and in vitro findings, and discuss an integrated mechanistic model of SHM.     

DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome

Poulogiannis G, Frayling I M & Arends M J
(2010) Histopathology 56, 167–179 DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome DNA mismatch repair (MMR) deficiency is one of the best understood forms of genetic instability in colorectal cancer (CRC), and is characterized by the loss of function of the MMR pathway. Failure to repair replication‐associated errors due to a defective MMR system allows persistence of mismatch mutations all over the genome, but especially in regions of repetitive DNA known as microsatellites, giving rise to the phenomenon of microsatellite instability (MSI). A high frequency of instability at microsatellites (MSI‐H) is the hallmark of the most common form of hereditary susceptibility to CRC, known as Lynch syndrome (LS) (previously known as hereditary non‐polyposis colorectal cancer syndrome), but is also observed in ～15–20% of sporadic colonic cancers (and rarely in rectal cancers). Tumour analysis by both MMR protein immunohistochemistry and DNA testing for MSI is necessary to provide a comprehensive picture of molecular abnormality, for use in conjunction with family history data and other clinicopathological features, in order to distinguish LS from sporadic MMR‐deficient CRC. Identification of the gene targets that become mutated in MMR‐deficient tumours may explain, at least in part, some of the clinical, pathological and biological features of MSI‐H CRCs and holds promise for developing novel therapeutics.     

Plasmodium falciparum MLH is schizont stage specific endonuclease

Malaria is one of the most important infectious diseases in many regions around the world including India. Plasmodium falciparum is the cause of most lethal form of malaria while Plasmodium vivax is the major cause outside Africa. Regardless of considerable efforts over the last many years there is still no commercial vaccine against malaria and the disease is mainly treated using a range of established drugs. With time, the malaria parasite is developing drug resistance to most of the commonly used drugs. This drug resistance might be due to defective mismatch repair in the parasite. Previously we have reported that the P. falciparum genome contains homologues to most of the components of mismatch repair (MMR) complex. In the present study we report the detailed biochemical characterization of one of the main component of MMR complex, MLH, from P. falciparum. Our results show that MLH is an ATPase and it can incise covalently closed circular DNA in the presence of Mn(2+) or Mg(2+) ions. Using the truncated derivatives we show that full length protein MLH is required for all the enzymatic activities. Using immunodepletion assays we further show that the ATPase and endomuclease activities are attributable to PfMLH protein. Using immunofluorescence assay we report that the peak expression of MLH in both 3D7 and Dd2 strains of P. falciparum is mainly in the schizont stages of the intraerythrocytic development, where DNA replication is active. MMR also contributes to the overall fidelity of DNA replication and the peak expression of MLH in the schizont stages suggests that MLH is most likely involved in correcting the mismatches occurring during replication. This study should make a significant contribution in our better understanding of DNA metabolic processes in the parasite.     

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.     

Pms2 is a genetic enhancer of trinucleotide CAG.CTG repeat somatic mosaicism: implications for the mechanism of triplet repeat expansion

The expansion of CAG.CTG repeat sequences is the cause of several inherited human disorders. Longer alleles are associated with an earlier age of onset and more severe symptoms, and are highly unstable in the germline and soma with a marked tendency towards repeat length gains. Germinal expansions underlie anticipation; whereas age-dependent, tissue-specific, expansion-biased somatic instability probably contributes toward the progressive nature and tissue-specificity of the symptoms. The mechanism(s) of repeat instability is not known, but recent data have implicated mismatch-repair (MMR) gene mutS homologues in driving expansion. To gain further insight into the expansion mechanism, we have determined the levels of somatic mosaicism of a transgenic expanded CAG.CTG repeat in mice deficient for the Pms2 MMR gene. Pms2 is a MutL homologue that plays a critical role in the downstream processing of DNA mismatches. The rate of somatic expansion was reduced by approximately 50% in Pms2-null mice. A higher frequency of rare, but very large, deletions was also detected in these animals. No significant differences were observed between Pms2(+/+) and Pms2(+/-) mice, indicating that a single functional Pms2 allele is sufficient to generate normal levels of somatic mosaicism. These findings reveal that as well as MMR enzymes that directly bind mismatched DNA, proteins that are subsequently recruited to the complex also play a central role in the accumulation of repeat length changes. These data suggest that somatic expansion results not by replication slippage, single stranded annealing or simple MutS-mediated stabilization of secondary structures, but by inappropriate DNA MMR.     

Inhibition of the 5' to 3' exonuclease activity of hEXO1 by 8-oxoguanine

The mismatch repair pathway is responsible for maintaining genomic stability by correcting base-base mismatches and insertion/deletion loops that arise mainly via replication errors. Additionally, the mismatch repair pathway performs a central role in the cellular response to both alkylation and reactive oxygen species induced DNA damage. An important step in mismatch processing is the recruitment of hEXO1, a 5' to 3' exonuclease, by hMSH2-hMSH6 to remove the nascent DNA strand. However, very little is currently known about the capacity of hEXO1 to exonucleolytically process damaged DNA bases. Therefore, we examined whether hEXO1 can degrade double-stranded DNA substrates containing alkylated or oxidized nucleotides. Our results demonstrated that hEXO1 is capable of degrading duplex DNA containing an O6-methylguanine (O6-meG) adduct paired with either a C or a T. Additionally, the hMSH2-hMSH6 complex stimulated hEXO1 exonuclease activity on the O6-meG/T and O6-meG/C DNA substrates. In contrast, hEXO1 exonuclease activity was significantly blocked by the presence of an 8-oxoguanine adduct in both single and double stranded DNA substrates. Further, hMSH2-hMSH6 was not able to alleviate the nucleolytic block caused by the 8-oxoguanine adduct in heteroduplex DNA.