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.     

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.     

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。     

DNA错配修复和临床肿瘤新进展

DNA复制是一个严谨有序的过程,细胞分裂时,碱基错配的概率约为1/1010～1/109.错配修复系统(MMR)是一个从细菌到真核细胞皆保守的DNA修复途径,它负责修复DNA中错配的碱基,或者DNA聚合酶的校对功能失调而引起的复制错误,插入,遗失碱基,使DNA整体复制的保真度增加50～1000倍.MMR系统失控会导致DN...     

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。     

微卫星DNA不稳定性及其在星形细胞肿瘤中的意义

星形细胞肿瘤中微卫星DNA不稳定性的出现及其频率取决于肿瘤的恶性程度.恶性程度高的星形细胞肿瘤常可检出MSI,且伴有错配修复基因的突变率也较高.某些MSI位点可作为间变性星形细胞瘤预后的一个指征.由于某些错配修复基因的缺陷导致微卫星DNA不稳定性,基因组不稳定性的影响在星形细胞肿瘤的发生、发展过程中具有重要意义.     

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ＭｕｔＨＬＳ修复系统，酵母中的错配修复基因及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，防止自由突变以及肿瘤的发生发展、诊断和治疗有着重要意义。     

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。     

揭示嵌入基因组DNA中RNA移除机制

据Shen Y 2011年12月4日（Nat Struct Mol Biol,2011 Dec 4.doi：10.1038/nsmb.2176）报道,核糖核酸酶H（RNase H）和DNA错配修复系统,似乎相互作用将DNA中RNA组分清除掉。RNA组分单元嵌入到含有一个有机体全部遗传数据的基因组DNA时,它们能够导致细胞产生问题。     

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.     

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.     

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.     

Role of DNA mismatch repair in apoptotic responses to therapeutic agents

Deficiencies in DNA mismatch repair (MMR) have been found in both hereditary cancer (i.e., hereditary nonpolyposis colorectal cancer) and sporadic cancers of various tissues. In addition to its primary roles in the correction of DNA replication errors and suppression of recombination, research in the last 10 years has shown that MMR is involved in many other processes, such as interaction with other DNA repair pathways, cell cycle checkpoint regulation, and apoptosis. Indeed, a cell's MMR status can influence its response to a wide variety of chemotherapeutic agents, such as temozolomide (and many other methylating agents), 6-thioguanine, cisplatin, ionizing radiation, etoposide, and 5-fluorouracil. For this reason, identification of a tumor's MMR deficiency (as indicated by the presence of microsatellite instability) is being utilized more and more as a prognostic indicator in the clinic. Here, we describe the basic mechanisms of MMR and apoptosis and investigate the literature examining the influence of MMR status on the apoptotic response following treatment with various therapeutic agents. Furthermore, using isogenic MMR-deficient (HCT116) and MMR-proficient (HCT116 3-6) cells, we demonstrate that there is no enhanced apoptosis in MMR-proficient cells following treatment with 5-fluoro-2'-deoxyuridine. In fact, apoptosis accounts for only a small portion of the induced cell death response.     

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.     

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.     

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.     

Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition

Mutational heterogeneity must be taken into account when reconstructing evolutionary histories, calibrating molecular clocks, and predicting links between genes and disease. Selective pressures and various DNA transactions have been invoked to explain the heterogeneous distribution of genetic variation between species, within populations, and in tissue-specific tumors. To examine relationships between such heterogeneity and variations in leading- and lagging-strand replication fidelity and mismatch repair, we accumulated 40,000 spontaneous mutations in eight diploid yeast strains in the absence of selective pressure. We found that replicase error rates vary by fork direction, coding state, nucleosome proximity, and sequence context. Further, error rates and DNA mismatch repair efficiency both vary by mismatch type, responsible polymerase, replication time, and replication origin proximity. Mutation patterns implicate replication infidelity as one driver of variation in somatic and germline evolution, suggest mechanisms of mutual modulation of genome stability and composition, and predict future observations in specific cancers.DNA synthesis errors are a dual-edged sword. At a population level, accurate DNA replication maintains species identity, yet a small fraction of replication errors creates mutations that improve fitness and fuel evolution. At an individual level, DNA synthesis errors can be beneficial, e.g., by allowing a virus or microbe to survive in an adverse environment or by promoting affinity maturation of antibodies. Replication errors can also result in mutations that have deleterious consequences, cell death, or carcinogenesis. Because replication fidelity underpins so much biology, it has been intensively studied. These studies reveal that—in the absence of stress—replication fidelity is largely determined by nucleotide selectivity, proofreading, and mismatch repair (MMR), with considerable heterogeneity in each process (for review, see Kunkel 2009). Mutation rate heterogeneity is a feature of evolution (Sasaki et al. 2009; Prendergast and Semple 2011; Tolstorukov et al. 2011), including somatic evolution, i.e., tumorigenesis (for review, see Salk et al. 2010). This heterogeneity complicates the identification of genes responsible for the initiation and progression of cancer (Lawrence et al. 2013). Our understanding of the origins of heterogeneous replication fidelity is limited because most studies only monitor a tiny fraction of large, highly organized genomes. Whole-genome studies are required for a complete picture of variations in replication fidelity, the underlying mechanisms, and the consequences for evolution and disease.One way to interrogate global replication fidelity is to allow mutations to accumulate through many cell divisions with minimal selection against deleterious mutations, and then to sequence the genome to identify the types, numbers, and locations of the mutations that arise (Nishant et al. 2009). To focus on replication errors per se, rather than on other sources of spontaneous mutations, mutation accumulation can be studied in cells defective in nucleotide selectivity, proofreading, or MMR. Such studies have been done in Saccharomyces cerevisiae, whose haploid nuclear genome contains 16 chromosomes and 12 million base pairs (bp). Studies of strains with complete or partial defects in MMR reported the accumulation of 76 to 140 mutations, mostly deletions in homonucleotide runs (Zanders et al. 2010; Ma et al. 2012; Lang et al. 2013). Another study of MMR-deficient haploid yeast (Serero et al. 2014) reported 1679 mutations, mostly substitutions. We (Larrea et al. 2010) previously used an MMR-defective haploid strain encoding a mutator variant of DNA polymerase delta (Pol delta), one of three major nuclear replicases. From the genome-wide distribution of 1099 transitions that accumulated and from similar studies using a reporter gene (for review, see Kunkel and Burgers 2008; Lujan et al. 2013), we proposed a model wherein DNA polymerase alpha (Pol alpha) and Pol delta are primarily lagging-strand replicases, whereas polymerase epsilon (Pol epsilon) is primarily a leading-strand replicase.In these studies, small data sets and/or selective pressures precluded correlation of mutations with other key features of genomic structure. Here we report a study based on more than 40,000 mutations, accumulated in the absence of selective pressure, in diploid yeast encoding wild-type replicases or mutator variants of Pol alpha, delta, or epsilon, each either proficient or defective in MMR. The results allow calculations of single-base error rates per base pair per generation for replication across the yeast nuclear genome. They also permit genome-wide estimates of the efficiency of MMR for different mismatches. We find that fidelity varies with DNA sequence context, and establish relationships between fidelity and replication origins, replication timing, nucleosome positions, and protein coding potential.