DNA mismatch repair,microsatellite instability and cancer

Mismatch (MMR) repair system plays a significant role in restoration of stability in the genome. Mutations in mismatch repair genes hamper their activity thus bring about a defect in mismatch repair (MMR) mechanism thereby conferring instability in the microsatellite sequences of both the coding and non-coding regions of the genome. Mutated mismatch repair genes result in the expansion or contraction of microsatellite sequence and confer microsatellite unstable or replication error positive phenotype. Hypermethylation of promoter regions of some of the MMR genes also causes inactivation of these genes and thus contribute to MSI. Microsatellite instability is an indicator of MMR deficiency and is a prime cause of varied tumorogenesis.     

DNA mismatch repair in trinucleotide repeat instability

Trinucleotide repeat expansions cause over 30 severe neuromuscular and neurodegenerative disorders, including Huntington's disease, myotonic dystrophy type 1, and fragile X syndrome. Although previous studies have substantially advanced the understanding of the disease biology, many key features remain unknown. DNA mismatch repair(MMR) plays a critical role in genome maintenance by removing DNA mismatches generated during DNA replication. However, MMR components,particularly mismatch recognition protein MutSβ and its interacting factors MutLα and MutLγ, have been implicated in trinucleotide repeat instability. In this review, we will discuss the roles of these key MMR proteins in promoting trinucleotide repeat instability.     

Human mismatch repair, drug-induced DNA damage, and secondary cancer

DNA mismatch repair (MMR) is an important replication error avoidance mechanism that prevents mutation. The association of defective MMR with familial and sporadic gastrointestinal and endometrial cancer has been acknowledged for some years. More recently, it has become apparent that MMR defects are common in acute myeloid leukaemia/myelodysplastic syndrome (AML/MDS) that follows successful chemotherapy for a primary malignancy. Therapy-related haematological malignancies are often associated with treatment with alkylating agents. Their frequency is increasing and they now account for at least 10% of all AML cases. There is also evidence for an association between MMR deficient AML/MDS and immunosuppressive treatment with thiopurine drugs. Here we review how MMR interacts with alkylating agent and thiopurine-induced DNA damage and suggest possible ways in which MMR defects may arise in therapy-related AML/MDS.     

DNA repair: models for damage and mismatch recognition

Maintaining the integrity of the genome is critical for the survival of any organism. To achieve this, many families of enzymatic repair systems which recognize and repair DNA damage have evolved. Perhaps most intriguing about the workings of these repair systems is the actual damage recognition process. What are the chemical characteristics which are common to sites of nucleic acid damage that DNA repair proteins may exploit in targeting sites? Importantly, thermodynamic and kinetic principles, as much as structural factors, make damage sites distinct from the native DNA bases, and indeed, in many cases, these are the features which are believed to be exploited by repair enzymes. Current proposals for damage recognition may not fulfill all of the demands required of enzymatic repair systems given the sheer size of many genomes, and the efficiency with which the genome is screened for damage. Here we discuss current models for how DNA damage recognition may occur and the chemical characteristics, shared by damaged DNA sites, of which repair proteins may take advantage. These include recognition based upon the thermodynamic and kinetic instabilities associated with aberrant sites. Additionally, we describe how small changes in base pair structure can alter also the unique electronic properties of the DNA base pair pi-stack. Further, we describe photophysical, electrochemical, and biochemical experiments in which mismatches and other local perturbations in structure are detected using DNA-mediated charge transport. Finally, we speculate as to how this DNA electron transfer chemistry might be exploited by repair enzymes in order to scan the genome for sites of damage.     

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.     

Disease-associated repeat instability and mismatch repair

Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential.     

DNA mismatch repair: the hands of a genome guardian

DNA mismatch repair (MMR) is initiated when the MutS protein recognizes damaged DNA. Crystal structures of MutS bound to mispaired and unpaired DNA show how MutS distinguishes damaged from undamaged DNA and explain how a broad variety of DNA mismatch lesions can be detected. The structures suggest mechanisms for the ATP-induced structural regulation of multistep DNA repair processes.     

Role of the mismatch repair gene, Msh6, in suppressing genome instability and radiation-induced mutations

Mismatch repair (MMR) is critical for preserving genomic integrity. Failure of this system can accelerate somatic mutation and increase the risk of developing cancer. MSH6, in complex with MSH2, is the MMR protein that mediates DNA repair through the recognition of 1- and 2-bp mismatches. To evaluate the effects of MSH6 deficiency on genomic stability we compared the frequency of in vivo loss of heterozygosity (LOH) between MSH6-proficient and deficient, 129S2xC57BL/6 F1 hybrid mice that were heterozygous for our reporter gene Aprt. We recovered mutant cells that had functionally lost APRT protein activity and categorized the spectrum of mutations responsible for the LOH events. We also measured the mutant frequency at the X-linked gene, Hprt, as a second reporter for point mutation. In Msh6-/-Aprt+/- mice, mutation frequency at Aprt was elevated in both T cells and fibroblasts by 2.5-fold and 5.7-fold, respectively, over Msh6+/+Aprt+/- littermate controls. While a modest increase in mitotic recombination (MR) was observed in MSH6-deficient fibroblasts compared to wild type controls, point mutation was the predominant mechanism leading to APRT deficiency in both cell types. Base substitution, consisting of multiple types of transitions, accounted for all of the point mutations identified within the Aprt coding region. We also assessed the role of MSH6 in preventing mutations caused by a common environmental mutagen, ionizing radiation (IR). In Msh6-/-Aprt+/- mice, 4Gy of X-irradiation induced a significant increase in point mutations at both Aprt and Hprt in T cells, but not in fibroblasts. These findings indicate that MutS alpha reduces spontaneous and IR-induced mutation in a cell type-dependant manner.     

Brc1-mediated DNA repair and damage tolerance

The structural maintenance of chromosome (SMC) proteins are key elements in controlling chromosome dynamics. In eukaryotic cells, three essential SMC complexes have been defined: cohesin, condensin, and the Smc5/6 complex. The latter is essential for DNA damage responses; in its absence both repair and checkpoint responses fail. In fission yeast, the UV-C and ionizing radiation (IR) sensitivity of a specific hypomorphic allele encoding the Smc6 subunit, rad18-74 (renamed smc6-74), is suppressed by mild overexpression of a six-BRCT-domain protein, Brc1. Deletion of brc1 does not result in a hypersensitivity to UV-C or IR, and thus the function of Brc1 relative to the Smc5/6 complex has remained unclear. Here we show that brc1Delta cells are hypersensitive to a range of radiomimetic drugs that share the feature of creating lesions that are an impediment to the completion of DNA replication. Through a genetic analysis of brc1Delta epistasis and by defining genes required for Brc1 to suppress smc6-74, we find that Brc1 functions to promote recombination through a novel postreplication repair pathway and the structure-specific nucleases Slx1 and Mus81. Activation of this pathway through overproduction of Brc1 bypasses a repair defect in smc6-74, reestablishing resolution of lesions by recombination.     

hMRE11 deficiency leads to microsatellite instability and defective DNA mismatch repair

DNA mismatch repair (MMR) is essential in the surveillance of accurate transmission of genetic information, and defects in this pathway lead to microsatellite instability and hereditary nonpolyposis colorectal cancer (HNPCC). Our previous study raised the possibility that hMRE11 might be involved in MMR through physical interaction with hMLH1. Here, we show that hMRE11 deficiency leads to significant increase in MSI for both mono- and dinucleotide sequences. Furthermore, RNA-interference-mediated hMRE11-knockdown in HeLa cells results in MMR deficiency. Analysis of seven HNPCC-associated hMLH1 missense mutations located within the hMRE11-interacting domain shows that four mutations (L574P, K618T, R659P and A681T) cause near-complete disruption of the interaction between hMRE11 and hMLH1, and two mutations (Q542L and L582V) cause a 30% reduction of protein interaction. These findings indicate that hMRE11 represents a functional component of the MMR pathway and the disruption of hMLH1-hMRE11 interaction could be an alternative molecular explanation for hMLH1 mutations in a subset of HNPCC tumours.     

Nuclear reorganization of DNA mismatch repair proteins in response to DNA damage

The DNA mismatch repair (MMR) system is highly conserved and vital for preserving genomic integrity. Current mechanistic models for MMR are mainly derived from in vitro assays including reconstitution of strand-specific MMR and DNA binding assays using short oligonucleotides. However, fundamental questions regarding the mechanism and regulation in the context of cellular DNA replication remain. Using synchronized populations of HeLa cells we demonstrated that hMSH2, hMLH1 and PCNA localize to the chromatin during S-phase, and accumulate to a greater extent in cells treated with a DNA alkylating agent. In addition, using small interfering RNA to deplete hMSH2, we demonstrated that hMLH1 localization to the chromatin is hMSH2-dependent. hMSH2/hMLH1/PCNA proteins, when associated with the chromatin, form a complex that is greatly enhanced by DNA damage. The DNA damage caused by high doses of alkylating agents leads to a G₂ arrest after only one round of replication. In these G₂-arrested cells, an hMSH2/hMLH1 complex persists on chromatin, however, PCNA is no longer in the complex. Cells treated with a lower dose of alkylating agent require two rounds of replication before cells arrest in G₂. In the first S-phase, the MMR proteins form a complex with PCNA, however, during the second S-phase PCNA is missing from that complex. The distinction between these complexes may suggest separate functions for the MMR proteins in damage repair and signaling. Additionally, using confocal immunofluorescence, we observed a population of hMSH6 that localized to the nucleolus. This population is significantly reduced after DNA damage suggesting that the protein is shuttled out of the nucleolus in response to damage. In contrast, hMLH1 is excluded from the nucleolus at all times. Thus, the nucleolus may act to segregate a population of hMSH2–hMSH6 from hMLH1–hPMS2 such that, in the absence of DNA damage, an inappropriate response is not invoked.     

Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems.

Nucleotide excision repair and the long-patch mismatch repair systems correct abnormal DNA structures arising from DNA damage and replication errors, respectively. DNA synthesis past a damaged base (translesion replication) often causes misincorporation at the lesion site. In addition, mismatches are hot spots for DNA damage because of increased susceptibility of unpaired bases to chemical modification. We call such a DNA lesion, that is, a base damage superimposed on a mismatch, a compound lesion. To learn about the processing of compound lesions by human cells, synthetic compound lesions containing UV photoproducts or cisplatin 1,2-d(GpG) intrastrand cross-link and mismatch were tested for binding to the human mismatch recognition complex hMutS alpha and for excision by the human excision nuclease. No functional overlap between excision repair and mismatch repair was observed. The presence of a thymine dimer or a cisplatin diadduct in the context of a G-T mismatch reduced the affinity of hMutS alpha for the mismatch. In contrast, the damaged bases in these compound lesions were excised three- to fourfold faster than simple lesions by the human excision nuclease, regardless of the presence of hMutS alpha in the reaction. These results provide a new perspective on how excision repair, a cellular defense system for maintaining genomic integrity, can fix mutations under certain circumstances.     

Eukaryotic DNA mismatch repair

Eukaryotic mismatch repair (MMR) has been shown to require two different heterodimeric complexes of MutS-related proteins: MSH2-MSH3 and MSH2-MSH6. These two complexes have different mispair recognition properties and different abilities to support MMR. Alternative models have been proposed for how these MSH complexes function in MMR. Two different heterodimeric complexes of MutL-related proteins, MLH1-PMS1 (human PMS2) and MLH1-MLH3 (human PMS1) also function in MMR and appear to interact with other MMR proteins including the MSH complexes and replication factors. A number of other proteins have been implicated in MMR, including DNA polymerase delta, RPA (replication protein A), PCNA (proliferating cell nuclear antigen), RFC (replication factor C), Exonuclease 1, FEN1 (RAD27) and the DNA polymerase delta and epsilon associated exonucleases. MMR proteins have also been shown to function in other types of repair and recombination that appear distinct from MMR. MMR proteins function in these processes in conjunction with components of nucleotide excision repair (NER) and, possibly, recombination.     

DNA mismatch repair and cancer

Five human DNA mismatch repair genes have been identified that, when mutated, cause susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC). Mutational inactivation of both copies of a DNA mismatch repair gene results in a profound repair defect and progressive accumulation of mutations throughout the genome. Some of the mutations confer selective advantage on the cells, giving rise to cancer. Recent discoveries suggest that apart from postreplication repair, DNA mismatch repair proteins have several other functions that are highly relevant to carcinogenesis. These include DNA damage surveillance, prevention of recombination between nonidentical sequences and participation in meiotic processes (chromosome pairing). A brief overview of these different features of the human DNA mismatch repair system will be provided, with the emphasis in their implications in cancer development.