Human Pluripotent Stem Cells Have a Novel Mismatch Repair-dependent Damage Response

Human pluripotent stem cells (PSCs) are presumed to have robust DNA repair pathways to ensure genome stability. PSCs likely need to protect against mutations that would otherwise be propagated throughout all tissues of the developing embryo. How these cells respond to genotoxic stress has only recently begun to be investigated. Although PSCs appear to respond to certain forms of damage more efficiently than somatic cells, some DNA damage response pathways such as the replication stress response may be lacking. Not all DNA repair pathways, including the DNA mismatch repair (MMR) pathway, have been well characterized in PSCs to date. MMR maintains genomic stability by repairing DNA polymerase errors. MMR is also involved in the induction of cell cycle arrest and apoptosis in response to certain exogenous DNA-damaging agents. Here, we examined MMR function in PSCs. We have demonstrated that PSCs contain a robust MMR pathway and are highly sensitive to DNA alkylation damage in an MMR-dependent manner. Interestingly, the nature of this alkylation response differs from that previously reported in somatic cell types. In somatic cells, a permanent G₂/M cell cycle arrest is induced in the second cell cycle after DNA damage. The PSCs, however, directly undergo apoptosis in the first cell cycle. This response reveals that PSCs rely on apoptotic cell death as an important defense to avoid mutation accumulation. Our results also suggest an alternative molecular mechanism by which the MMR pathway can induce a response to DNA damage that may have implications for tumorigenesis.     

Mismatch repair (MMR) efficiency and <Emphasis Type="Italic">MSH2</Emphasis> gene mutation in human colorectal carcinoma cell line COLO320HSR

Colorectal cancer (CC) is one of two diseases, in which the link between cancer proneness and DNA repair deficiency appears to be proved. A strict relationship between mismatch repair (MMR) gene mutations, microsatellite instability (MSI) has been found in familiar colorectal cancer (Lynch syndrome). Tumorigenesis at familiar cancer is initiated by biallelic mutations in the major MMR genes, namely MSH2 or MLH1. One of these mutations is an inherited germline alteration and the other is a somatic one. The initiating mutation in sporadic colorectal tumors was not still identified although biochemical and genetic signs of MMR deficiency are observed in tumor cells. Two currently used colorectal tumor cell lines HCT116 and COLO320HSR were derived from hereditary and sporadic tumors accordingly. HCT116 cell line exhibits MMR-deficiency due to biallelic deletion in MLH1. As a consequence this shows MSI phenotype and a near-diploid karyotype. COLO320HSR cell line is characterized by MSS phenotype with mostly imbalanced aberrations. This indicates MMR proficiency in these cells. However, both MMR-deficient HCT116 and COLO320HSR cells reveal near-diploid karyotype. Earlier we have shown that the number of secondary DNA double strand breaks, induced by methylnitrosourea (MNU), represent functional activity of cellular MMR. In the present study, using this approach we evaluated sensitivity to MNU and MMR activity in two colorectal tumor cell lines (HCT116, COLO320HSR) and compared them to that in the HeLa cell line, which have MMR-proficient phenotype. We showed that cell line COLO320HSR exhibits low MMR activity, close to the level of MMR-activity in HCT116 cell line. We found a mutation in MSH2-G520A gene in COLO320HSR. This neutral mutation apparently is not related to polymorphism as we failed to identify the same mutation in any of MSH2 gene sequences of lymphocytes from 30 patients with sporadic colorectal cancer.     

Rad9 plays an important role in DNA mismatch repair through physical interaction with MLH1

Rad9 is conserved from yeast to humans and plays roles in DNA repair (homologous recombination repair, and base-pair excision repair) and cell cycle checkpoint controls. It has not previously been reported whether Rad9 is involved in DNA mismatch repair (MMR). In this study, we have demonstrated that both human and mouse Rad9 interacts physically with the MMR protein MLH1. Disruption of the interaction by a single-point mutation in Rad9 leads to significantly reduced MMR activity. This disruption does not affect S/M checkpoint control and the first round of G₂/M checkpoint control, nor does it alter cell sensitivity to UV light, gamma rays or hydroxyurea. Our data indicate that Rad9 is an important factor in MMR and carries out its MMR function specifically through interaction with MLH1.     

Mismatch repair as a source of mutations in non-dividing cells

This paper describes a mechanism which permits somatic cells to generate random mutations in the complete absence of cell proliferation. The mechanism itself is remarkably simple, involving a well-known cellular process (mismatch repair or MMR) which is primarily associated with mutation avoidance, but which is also capable of generating mutations when circumstances are not ideal for avoidance. When MMR operates in its so-called methylation-instructed mode to remove mismatches from newly-replicated portions of genomic DNA, it does so in a way which serves to minimize mutation yields. By contrast, when MMR operates in a non-instructed or randomly-templated way to remove mismatches from DNA molecules, it does so without distinguishing between the two strands of DNA that contain the mismatched bases. Randomly-templated mismatch repair (RT-MMR) therefore generates new and complete mutations whenever it removes the correct bases from either base-pair mismatches or frameshift mispairs and replaces them without incorrect bases or sequences. Wider recognition of the existence of this mechanism — and especially of its proclivity for mutation generation when it is operating in non-dividing cells—should help us to develop a better understanding of a number of important biological phenomena, and may be of particular value in our attempts to explain the origins of many human cancers.     

Mutations in the nucleotide-binding domain of MutS homologs uncouple cell death from cell survival

After genotoxic insult, the decision to repair or undergo cell death is pivotal for undamaged cell survival, and requires a highly controlled coordination of both pathways. Disruption of this regulation results in tumorigenesis and failure of cancer therapy. Mismatch repair (MMR) proteins have a unique role by contributing to both pathways, though direct evidence for their function in the DNA damage response is ambiguous. We report separation of function mutants in the ATPase domains of yeast MutS homologous (MSH) proteins that uncouple MMR-dependent DNA repair from damage response to cisplatin. While mutations in the ATPase domain have devastating effects on the mutation rate of the cell, ATPase processing is mostly dispensable for the cell death phenotype; only limited processing by the MSH6 subunit is required in DNA damage response. Different DNA binding patterns and nucleotide sensitivity of Msh2/Msh6-DNA adduct and protein-mismatch complexes, respectively, suggest that the presence of different DNA lesions influences the requirement for ATP. Limited proteolysis of purified protein gives first indications for differences in nucleotide-induced conformational changes in the presence of platinated DNA. Structural modeling of bacterial MutS proteins reinforces nucleotide-dependent differences in structures that contribute to the distinction between DNA damage response and repair. Our results demonstrate the uncoupling of MMR-dependent damage response from repair and present first indications for the involvement of distinct conformational changes in MSH proteins in this process. These data present evidence for a mechanism of MMR-dependent damage response that differs from MMR; these results have strong implications for the chemotherapeutic treatment of MMR-defective tumors.     

Dark-Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light III. Effect of rec Mutations on Recovery of Excision-Deficient Mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 unable to excise pyrimidine dimers from their deoxyribonucleic acid (DNA) because of a uvr mutation show a higher survival when plated on a minimal salts medium after exposure to ultraviolet radiation than when plated on a complex medium such as nutrient agar containing yeast extract. This response has been called minimal medium recovery (MMR). Recovery of uvr mutants can take place in liquid as well as on solid medium, but not in buffer or under conditions of amino acid starvation that do not permit cell growth and normal DNA replication. MMR can thus be distinguished from the recovery of recombination-deficient (rec(-)uvr(+)) derivatives of K-12 which can occur under conditions where growth is not possible. Because MMR is characteristic of excision-defective mutants, it evidently reflects a type of repair independent of excision. We have obtained genetic evidence that MMR is determined by the rec genes, which also control recombination in K-12. Cells carrying a uvr mutation together with recA13, recA56, recB21, or recC22 failed to show MMR and were more sensitive to ultraviolet radiation than either their rec(+)uvr(-) or rec(-)uvr(+) parents. The rec(+)uvr(-) derivatives obtained from recA uvr(-) strains by transduction or by reversion regained the capacity for MMR. Our results indicate that inactivation of any one of the three genes, recA, recB, or recC, prevents cells from showing MMR.     

Isolation and characterization of new proliferating cell nuclear antigen (POL30) mutator mutants that are defective in DNA mismatch repair

A number of studies have suggested a role for proliferating cell nuclear antigen (PCNA) in DNA mismatch repair (MMR). However, the majority of mutations in the POL30 gene encoding PCNA that cause MMR defects also cause replication and other repair defects that contribute to the increased mutation rate caused by these mutations. Here, 20 new pol30 mutants were identified and screened for MMR and other defects, resulting in the identification of two mutations, pol30-201 and pol30-204, that appear to cause MMR defects but little if any other defects. The pol30-204 mutation altered an amino acid (C81R) in the monomer-monomer interface region and resulted in a partial general MMR defect and a defect in MSH2-MSH6 binding in vitro. The pol30-201 mutation altered an amino acid (C22Y) located on the surface of the PCNA trimer that slides over the DNA but did not cause a defect in MSH2-MSH6 binding in vitro. The pol30-201 mutation caused an intermediate mutator phenotype. However, the pol30-201 mutation caused almost a complete defect in the repair of AC and GT mispairs and only a small defect in the repair of a "+T" insertion, an effect similar to that caused by an msh6Delta mutation, indicating that pol30-201 primarily effects MSH6-dependent MMR. The chromosomal double mutant msh3-FF>AA msh6-FF>AA eliminating the conserved FF residues of the PCNA interacting motif of these proteins caused a small (<10%) defect in MMR but showed synergistic interactions with mutations in POL30, indicating that the FF>AA substitution may not eliminate PCNA interactions in vivo. These results indicate that the interaction between PCNA and MMR proteins is more complex than was previously appreciated.     

Mismatch repair (MMR) efficiency and MSH2 gene mutation in human colorectal carcinoma cell line COLO320HSR

Colorectal cancer (CC) is one of two diseases, in which the link between cancer proneness and DNA repair deficiency appears to be proved. A strict relationship between mismatch repair (MMR) gene mutations, microsatellite instability (MSI) has been found in familiar colorectal cancer (Lynch syndrome). Tumorigenesis at familiar cancer is initiated by biallelic mutations in the major MMR genes, namely MSH2 or MLH1. One of these mutations is an inherited germline alteration and the other is a somatic one. The initiating mutation in sporadic colorectal tumors was not still identified although biochemical and genetic signs of MMR deficiency are observed in tumor cells. Two currently used colorectal tumor cell lines HCT116 and COLO320HSR were derived from hereditary and sporadic tumors accordingly. HCT116 cell line exhibits MMR-deficiency due to biallelic deletion in MLHL. As a consequence this shows MSI phenotype and a near-diploid karyotype. COLO320HSR cell line is characterized by MSS phenotype with mostly imbalanced aberrations. This indicates MMR proficiency in these cells. However, both MMR-deficient HCT116 and COLO320HSR cells reveal near-diploid karyotype. Earlier we have shown that the number of secondary DNA double strand breaks, induced by methylnitrosourea (MNU), represent functional activity of cellular MMR. In the present study, using this approach we evaluated sensitivity to MNU and MMR activity in two colorectal tumor cell lines (HCT 116, COLO320HSR) and compared them to that in the HeLa cell line, which have MMR-proficient phenotype. We showed that cell line COLO320HSR exhibits low MMR activity, close to the level of MMR-activity in HCT116 cell line. We found a mutation in MSH2-G520A gene in COLO320HSR. This neutral mutation apparently is not related to polymorphism as we failed to identify the same mutation in any of MSH2 gene sequences of lymphocytes from 30 patients with sporadic colorectal cancer.     

Novel PMS1 alleles preferentially affect the repair of primer strand loops during DNA replication

Null mutations in DNA mismatch repair (MMR) genes elevate both base substitutions and insertions/deletions in simple sequence repeats. Data suggest that during replication of simple repeat sequences, polymerase slippage can generate single-strand loops on either the primer or template strand that are subsequently processed by the MMR machinery to prevent insertions and deletions, respectively. In the budding yeast Saccharomyces cerevisiae and mammalian cells, MMR appears to be more efficient at repairing mispairs comprised of loops on the template strand compared to loops on the primer strand. We identified two novel yeast pms1 alleles, pms1-G882E and pms1-H888R, which confer a strong defect in the repair of "primer strand" loops, while maintaining efficient repair of "template strand" loops. Furthermore, these alleles appear to affect equally the repair of 1-nucleotide primer strand loops during both leading- and lagging-strand replication. Interestingly, both pms1 mutants are proficient in the repair of 1-nucleotide loop mispairs in heteroduplex DNA generated during meiotic recombination. Our results suggest that the inherent inefficiency of primer strand loop repair is not simply a mismatch recognition problem but also involves Pms1 and other proteins that are presumed to function downstream of mismatch recognition, such as Mlh1. In addition, the findings reinforce the current view that during mutation avoidance, MMR is associated with the replication apparatus.     

Visualization of mosaicism in tissues of normal and mismatch-repair-deficient mice carrying a microsatellite-containing transgene

We tested for azoospermia factor (AZF) deletions 17 loci corresponding to AZF subintervals a-d in 17 cases of testicular tumors occurring in Finns. While DNA samples from 48 CEPH and 32 Finnish males showed no deletions, patients with testicular cancer displayed AZF deletion mosaicisms in various non-tumor tissues (13 cases) and specific deletion haplotypes in tumor tissues (10 cases). Two of the cases with AZF deletions were testicular non-Hodgkin lymphomas indicating that Y-microdeletions appear also in malignancies other than seminoma and non-seminoma tumors. In good agreement with this assumption, we detected one AZF deletion in normal cells from 1 of 5 HNPCC cases, heterozygous for an MLH1 mutation. We propose that AZF deletions occur in early embryogenesis due to mutations of TSPY, mismatch repair (MMR), or X-specific genes. Since fathers of testicular, tumor cases did not exhibit AZF deletions, we assumed they were not carriers of the mutation inducing AZF deletion-mosaicisms. Therefore, tumor cases should have received the MMR gene or X mutations via the maternal lineage, or for the case of TSPY and MMR genes via a sperm carrying a mutation occurred in the paternal germ-cell line. We consider AZF microdeletions in non-tumor cells to be part of a broader pattern of chromosome instability producing susceptibility to testicular tumors. Clonal transformation and expansion of one of these tumor-susceptible cell lineages give rise to testicular tumors showing genome anomalies characteristic of testicular cancers (i12p, LOH and genetic imbalance for various autosomal regions, Y- and autosomal MSI, specific AZF deletion haplotypes).     

Mismatch Repair Protein Deficiency Compromises Cisplatin-induced Apoptotic Signaling

Mismatch repair (MMR) proteins participate in cytotoxicity induced by certain DNA damage-inducing agents, including cisplatin (cis-diamminedichloroplatinum(II), CDDP), a cancer chemotherapeutic drug utilized clinically to treat a variety of malignancies. MMR proteins have been demonstrated to bind to CDDP-DNA adducts and initiate MMR protein-dependent cell death in cells treated with CDDP; however, the molecular events underlying this death remain unclear. As MMR proteins have been suggested to be important in clinical responses to CDDP, a clear understanding of MMR protein-dependent, CDDP-induced cell death is critical. In this report, we demonstrate MMR protein-dependent relocalization of cytochrome c to the cytoplasm and cleavage of caspase-9, caspase-3, and poly(ADP-ribose) polymerase upon treatment of cells with CDDP. Chemical inhibition of caspases specifically attenuates CDDP/MMR protein-dependent cytotoxicity, suggesting that a caspase-dependent signaling mechanism is required for the execution of this cell death. p53 protein levels were up-regulated independently of MMR protein status, suggesting that p53 is not a mediator of MMR-dependent, CDDP-induced death. This work is the first indication of a required signaling mechanism in CDDP-induced, MMR protein-dependent cytotoxicity, which can be uncoupled from other CDDP response pathways, and defines a critical contribution of MMR proteins to the control of cell death.The MMR² system of proteins plays roles in diverse cellular processes, perhaps most notably in preserving genomic integrity by recognizing and facilitating the repair of post-DNA replication base pairing errors. Recognition of these errors and recruitment of repair machinery is performed by the MutSα complex (consisting of the MMR proteins MSH2 and MSH6) or MutSβ complex (consisting of MSH2 and MSH3). Defects in MMR proteins render cells hypermutable and promote microsatellite instability, a hallmark of MMR defects. MMR protein defects are found in a wide variety of sporadic cancers, as well as in hereditary non-polyposis colorectal cancer (1).In addition to their role in DNA repair, MMR proteins also play a role in cytotoxicity induced by specific types of DNA-damaging chemotherapeutic drugs, such as CDDP, which is utilized clinically to treat a number of different cancer types. MutSα recognizes multiple types of DNA damage, including 1,2-intrastrand CDDP adducts and O⁶-methylguanine lesions (2). Treatment of cells with compounds that induce these types of lesions, including CDDP and methylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), results in MMR protein-dependent cell cycle arrest and cell death (3–7). This suggests that MMR proteins, in addition to their role in DNA repair, are also capable of initiating cell death in response to certain types of DNA damage.Cells treated with DNA-damaging agents frequently activate an apoptotic cell death pathway mediated by the mitochondria. This intrinsic death signaling pathway predominantly involves the coordinated activity of two groups of proteins: pro-death members of the Bcl-2 family that control the integrity of mitochondrial membranes, and members of the caspase family of cysteinyl proteases that proteolytically cleave intracellular substrates, giving rise to apoptotic morphology and destruction of the cell (8, 9). Pro-death Bcl-2 family members, such as Bax and Bak, target the outer mitochondrial membrane and cause the cytosolic release of pro-death factors residing within the mitochondria of unstressed cells (8). Predominant among these factors is cytochrome c, whose cytoplasmic localization results in the formation of a caspase-activating platform known as the apoptosome (10). This complex includes the adaptor protein Apaf-1, and when formed the apoptosome promotes the cleavage and activation of caspase-9 (11, 12). Once activated, this apical caspase proceeds to cleave and activate caspase-3, the predominant effector protease of apoptosis.A significant amount of evidence has been gathered illustrating MMR protein-dependent pro-death signaling in response to methylating agents (13–16, 3). In contrast, the MMR protein-dependent cytotoxic response to CDDP is largely unknown, with only the p53-related transactivator protein p73 and the c-Abl kinase clearly implicated as potential mediators of CDDP/MMR protein-dependent cell death in human cells (17, 18). Interestingly, ATM, Chk1, Chk2, and p53, which are activated in an MMR protein-dependent manner after treatment of cells with MNNG (3, 13), are not involved in the MMR-dependent response to CDDP (7, 17). In addition, the magnitude of MMR protein-dependent cell death induced by methylating agents and CDDP differs (4). These findings suggest that unique signaling pathways may be engaged by MMR proteins depending upon the type of recognized lesion. As such, there is a requirement for further study of the molecular events underlying MMR protein-dependent cell death and cell cycle arrest for each type of recognized DNA lesion. This is particularly relevant in the case of CDDP, as evidence from a limited number of retrospective clinical studies suggests that MMR proteins play an important role in patient response to CDDP. Several studies examining immunohistochemical staining against MSH2 or MLH1 have demonstrated that levels of these proteins are reduced in ovarian and esophageal tumor samples following CDDP-based chemotherapy (19, 20). Low levels of MMR protein post-chemotherapy seem to be predictive of lower overall survival in a certain subset of tumors (esophageal cancer), but not others (ovarian and non-small cell lung cancer) (19–21). Two recent studies examining MMR protein levels and microsatellite instability in germ cell tumors from patients receiving platinum-based chemotherapy have suggested a prognostic value for pre-chemotherapy MMR protein status in these tumors (22, 23). This potential clinical relevance underscores the need for a greater understanding of MMR protein-dependent mechanisms of CDDP-induced cell death.In this study, we report that CDDP induces an MMR protein-dependent decrease in cell viability and MMR protein-dependent signaling in the form of cytochrome c release to the cytoplasm and cleavage of caspase-9, caspase-3, and PARP. Chemical inhibition of caspases specifically attenuates CDDP/MMR protein-dependent loss of cell viability, indicating a requirement for caspase activation in this process and uncoupling MMR protein-dependent cytotoxic signaling from other CDDP response pathways. Additionally, the CDDP-induced, MMR protein-dependent cytotoxic response is independent of p53 signaling. Our results demonstrate for the first time an MMR protein-dependent pro-death signaling pathway in cells treated with CDDP.     

高频度微卫星不稳定性大肠癌"修饰型"微卫星变化与MLH1及KRAS基因突变密切相关

本研究通过方法学的改良和观察方式的创新试图阐明这种现象的原因.微卫星非传统的检测方法仅能实现微卫星定性检测,我所在的研究组开发了自动片段分析双荧光标识技术,提高了微卫星检测的感度和重复性,并实现了微卫星片段变化长度的定量.小于6碱基的微卫星变化被定义为修饰型微卫星不稳定,大于8碱基的变化被定义为跳跃型微卫星不稳定,它们的电泳谱截然不同.前者表现为在非肿瘤来源微卫星位点基础上的增加或减少,后者表现为距离非肿瘤微卫星片段远隔部位的新波形的出现.通过研究我们发现,在DNA错配修复缺陷细胞系及基因敲除大鼠自发肿瘤样本,仅有修饰型微卫星不稳定性检出;在人类DNA错配修复缺陷细胞系连续80次传代也没有检出跳跃型变化.跳跃型变化不能通过简单重复序列不稳定基础上的增加或减少的累加而获得.在76例散发大肠癌,我们检测了微卫星不稳定性,KRAS基因突变,并对高频度微卫星不稳定性病例的两个主要DNA错配修复基因MSH2和MLHl进行了全长测序.我们发现,在大肠癌,按频度的传统分类与按波形变化的分类有高度的一致性,高频度微卫星不稳定性病例均检测到跳跃型表现,低频度微卫星不稳定性都表现为修饰型变化.在12例高频度微卫星不稳定病例,有三例检出了跳跃型和修饰型同时存在微卫星不稳定的特殊表型,这3例均检出KRAS的突变,更有趣的是该3例病例也同时检出了DNA错配修复基因MLH1的变异.而在其他9例高频度微卫星不稳定病例,KRAS突变及MLH1、MSH2交变未检出.通过对突变谱的分析我们还发现,修饰型微卫星不稳定与KTAS基因12号密码子的转换型突变高度相关,而微卫星稳定的病例检出的KRAS基因12号密码子突变多为颠换型突变.修饰型微卫星不稳定表型检出的高频度转换突变可由DNA错配修复缺陷的分子背景解释.通过本研究,我们认为以波形为基础的微卫星不稳定新分型可能是解决目前微卫星研究领域矛盾的一个选项.一直公认为高频度微卫星不稳定性是"真正"的DNA错配修复缺陷表型,我们的研究提示实际上高频度微卫星的可能是多元的.修饰型微卫星不稳定与DNA错配修复缺陷直接关联,而跳跃型微卫星不稳定的原因尚未阐明.在高频度为微型不稳定中,携带修饰型变化的病例可以通过DNA错配修复系统缺陷来解释其病因.     

A model for initial DNA lesion recognition by NER and MMR based on local conformational flexibility

Initial recognition of DNA damage is the crucial but poorly understood first step in DNA repair by the human nucleotide excision repair(NER) and mismatch repair (MMR) systems. Failure by NER or MMR to recognize DNA damage threatens the genetic integrity of the organism and may play a role in carcinogenesis. Both NER and MMR recognize and repair a wide variety of structurally dissimilar lesions against the background of normal DNA. Previous studies have suggested that detection of thermodynamic destabilization of DNA caused by covalent damage and base mismatches is a potential mechanism by which repair pathways with broad specificity such as NER and MMR recognize their substrates. However, both NER and MMR respectively, repair a wide variety of stabilizing and destabilizing covalent DNA lesions and base pair mismatches. A common feature of lesions that are both thermodynamically stabilizing and destabilizing is the alteration of the local DNA flexibility (dynamics). In this review we describe the experimental evidence for altered dynamics from NMR and thermodynamic studies on normal and damaged DNA molecules with respect to recognition by NER and MMR. Based on these data, we propose a model for initial detection of lesions by both NER and MMR that occurs through an indirect readout mechanism of alternative DNA conformations induced by covalent damage and base mismatches.     

Endometrial Cancer as a Familial Tumor: Pathology and Molecular Carcinogenesis (Review)

Some cases of endometrial cancer are associated with a familial tumor and are referred to as hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Such tumors are thought to be induced by germline mutation of the DNA mismatch repair (MMR) gene, but many aspects of the pathology of familial endometrial cancer are unclear and no effective screening method has been established. However, the pathology of endometrial cancer with familial tumor has been progressively clarified in recent studies. At present, about 0.5% of all cases of endometrial cancers meet the clinical diagnostic criteria for HNPCC. A recent analysis of the three MMR genes (hMLH1, hMSH2 and hMSH6) revealed germline mutations in 18 of 120 cases (15.0%) of endometrial cancer with familial accumulation of cancer or double cancer, with a frameshift mutation of the hMSH6 gene being the most common. Many cases with mutation did not meet the current clinical diagnostic criteria for HNPCC, indicating that familial endometrial cancer is often not diagnosed as HNPCC. The results suggest that the hMSH6 gene mutation may be important in carcinogenesis in endometrial cancer and germline mutations of the MMR gene may be more prevalent in cases associated with familial accumulation of cancer. An international large-scale muticenter study is required to obtain further information about the pathology of endometrial cancer as a familial tumor.Key Words: HNPCC, Endometrial cancer, DNA mismatch repair gene, hMLH1, hMSH6.     

The cell cycle and DNA mismatch repair

The DNA mismatch repair (MMR) pathway contributes to the fidelity of DNA synthesis and recombination by correcting mispaired nucleotides and insertion/deletion loops (IDLs). We have investigated whether MMR protein expression, activity, and subcellular location are altered during discrete phases of the cell cycle in mammalian cells. Two distinct methods have been used to demonstrate that although physiological MMR protein expression, mismatch binding, and nick-directed MMR activity within the nucleus are at highest levels during S phase, MMR is active throughout the cell cycle. Despite equal MMR nuclear protein concentrations in S and G(2) phases, mismatch binding and repair activities within G(2) are significantly lower, indicating a post-translational decrease in MMR activity specific to G(2). We further demonstrate that typical co-localization of MutSalpha to late S phase replication foci can be disrupted by 2 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This concentration of MNNG does not decrease ongoing DNA synthesis nor induce cell cycle arrest until the second cell cycle, with long-term colony survival decreased by only 24%. These results suggest that low level alkylation damage can selectively disrupt MMR proofreading activity during DNA synthesis and potentially increase mutation frequency within surviving cells.     

The Escherichia coli methyl-directed mismatch repair system repairs base pairs containing oxidative lesions

A major role of the methyl-directed mismatch repair (MMR) system of Escherichia coli is to repair postreplicative errors. In this report, we provide evidence that MMR also acts on oxidized DNA, preventing mutagenesis. When cells deficient in MMR are grown anaerobically, spontaneous mutation frequencies are reduced compared with those of the same cells grown aerobically. In addition, we show that a dam mutant has an increased sensitivity to hydrogen peroxide treatment that can be suppressed by mutations that inactivate MMR. In a dam mutant, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted to single- and double-strand DNA breaks. Thus, base pairs containing oxidized bases will be converted to strand breaks if they are repaired by MMR. This is demonstrated by the increased peroxide sensitivity of a dam mutant and the finding that the sensitivity can be suppressed by mutations inactivating MMR. We demonstrate further that this repair activity results from MMR recognition of base pairs containing 8-oxoguanine (8-oxoG) based on the finding that overexpression of the MutM oxidative repair protein, which repairs 8-oxoG, can suppress the mutH-dependent increase in transversion mutations. These findings demonstrate that MMR has the ability to prevent oxidative mutagenesis either by removing 8-oxoG directly or by removing adenine misincorporated opposite 8-oxoG or both.     

Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos

S(N)1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O(6)-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in replication fork stalling and cell death. We used a somatic mutation detection assay to study the in vivo effects of alkylation damage on lethality and mutation frequency in developing zebrafish embryos. Consistent with the damage-sensing role of the MMR system, mutant embryos lacking the MMR enzyme MSH6 displayed lower lethality than wild-type embryos after exposure to ENU and MNU. In line with this, alkylation-induced somatic mutation frequencies were found to be higher in wild-type embryos than in the msh6 loss-of-function mutants. These mutations were found to be chromosomal aberrations that may be caused by chromosomal breaks that arise from stalled replication forks. As these chromosomal breaks arise at replication, they are not expected to be repaired by non-homologous end joining. Indeed, Ku70 loss-of-function mutants were found to be equally sensitive to ENU as wild-type embryos. Taken together, our results suggest that in vivo alkylation damage results in chromosomal instability and cell death due to aberrantly processed MMR-induced stalled replication forks.     

Neurofibromatosis type 1 gene as a mutational target in a mismatch repair-deficient cell type

DNA mismatch repair (MMR) is the process by which incorrectly paired DNA nucleotides are recognized and repaired. A germline mutation in one of the genes involved in the process may be responsible for a dominantly inherited cancer syndrome, hereditary nonpolyposis colon cancer. Cancer progression in predisposed individuals results from the somatic inactivation of the normal copy of the MMR gene, leading to a mutator phenotype affecting preferentially repeat sequences (microsatellite instability, MSI). Recently, we identified children with a constitutional deficiency of MMR activity attributable to a mutation in the h MLH1 gene. These children exhibited a constitutional genetic instability associated with clinical features of de novo neurofibromatosis type 1 (NF1) and early onset of extracolonic cancer. Based on these observations, we hypothesized that somatic NF1 gene mutation was a frequent and possibly early event in MMR-deficient cells. To test this hypothesis, we screened for NF1 mutations in cancer cells. Genetic alterations were identified in five out of ten tumor cell lines with MSI, whereas five MMR-proficient tumor cell lines expressed a wild-type NF1 gene. Somatic NF1 mutations were also detected in two primary tumors exhibiting an MSI phenotype. Finally, a 35-bp deletion in the murine Nf1 coding region was identified in mlh1-/- mouse embryonic fibroblasts. These observations demonstrate that the NF1 gene is a mutational target of MMR deficiency and suggest that its inactivation is an important step of the malignant progression of MMR-deficient cells.