The role of DNA mismatch repair in immunotherapy of human cancer

DNA mismatch repair (MMR) is an important pathway which helps to maintain genomic stability. Mutations in DNA MMR genes are found to promote cancer initiation and foster tumor progression. Deficiency or inactivation of MMR results in microsatellite instability (MSI) which triggers neoantigen generation and impairs tumor growth. Immunotherapies targeting MMR can increase the burden of neoantigens in tumor cells. While MSI has been regarded as an important predictor of sensitivity and drug resistance for immunotherapy-based strategies. Different approaches targeting genomic instability have been demonstrated to be promising in malignancies derived from different tissues. Underlying MMR deficiency-associated immunogenicity is important for improving the therapeutic efficacy of immunotherapies. In this review we provide an overview of the MMR systems, their role in tumorigenesis, drug resistance, prognostic significance and potential targets for therapeutic treatment in human cancers, especially in hematological malignancies.     

Acetyltransferase p300 regulates NBS1-mediated DNA damage response

The role of p300 in DNA damage response is unclear. To understand how ATM-dependent phosphorylation of p300 affects its function in response to DNA damage, we present evidence that S106 of p300, which is phosphorylated by ATM, regulates stability of NBS1 and recruitment into damaged DNA, possibly leading to regulation of DNA repair. Non-phosphorylatable p300 (S106A) destabilized NBS1 and decreased NBS1–p300 interaction. The recruitment of NBS1 into damaged DNA was impaired in the presence of S106A. Also, a dominant negative p300 lacking enzymatic activity induced destabilization of NBS1, suggesting that its acetyltransferase is required for NBS1 stability. These results indicate that phosphorylation of p300 can regulate NBS1-mediated DNA damage response, and that these events occur in an acetylation-dependent manner.

Structured summary

MINT-8058074, MINT-8058083: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by anti bait coimmunoprecipitation (MI:0006)MINT-8058111: p300 (uniprotkb:Q09472) and NBS1 (uniprotkb:O60934) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-8058657: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by two hybrid (MI:0018)MINT-8058093: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by anti tag coimmunoprecipitation (MI:0007)     

DNA base repair – recognition and initiation of catalysis

Endogenous DNA damage induced by hydrolysis, reactive oxygen species and alkylation modifies DNA bases and the structure of the DNA duplex. Numerous mechanisms have evolved to protect cells from these deleterious effects. Base excision repair is the major pathway for removing base lesions. However, several mechanisms of direct base damage reversal, involving enzymes such as transferases, photolyases and oxidative demethylases, are specialized to remove certain types of photoproducts and alkylated bases. Mismatch excision repair corrects for misincorporation of bases by replicative DNA polymerases. The determination of the 3D structure and visualization of DNA repair proteins and their interactions with damaged DNA have considerably aided our understanding of the molecular basis for DNA base lesion repair and genome stability. Here, we review the structural biochemistry of base lesion recognition and initiation of one-step direct reversal (DR) of damage as well as the multistep pathways of base excision repair (BER), nucleotide incision repair (NIR) and mismatch repair (MMR).     

Autophagy regulates DNA repair through SQSTM1/p62

Macroautophagy/autophagy is primarily a degradative pathway that clears malfunctioning cellular components in response to various types of stress. Recent studies have indicated that autophagy also plays an important role in maintaining genome stability. Loss of autophagy is associated with increased damage to DNA, inappropriate amplification of genomic regions and abnormal chromosome number. In a recent paper by Wang et al. the authors uncover a mechanism through which autophagy regulates the ubiquitination of chromatin. In particular, the autophagy receptor and substrate SQSTM1/p62 inhibits the E3 ligase RNF168-dependent ubiquitination of histone in response to DNA double-strand breaks. Dysregulation of this process leads to a reduced ability to repair DNA and a corresponding increase in the sensitivity of cells to radiation-induced damage.     

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

Base excision repair （BER） is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged （oxidized or aikylated） or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species （ROS）. BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease （APE） to generate 3＇ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA iigase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APEI, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3＇ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and iigases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organeile targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.     

重离子辐照酿酒酵母DNA损伤修复途径研究进展

重离子辐照通过直接和间接作用导致生物体DNA产生损伤,包括DNA的链断裂、碱基的插入或丢失以及氧化损伤等.DNA损伤直接影响复制、转录和蛋白质合成,同时还是突变的重要原因,因此,DNA损伤修复系统尤为重要.在酿酒酵母中,这些损伤主要是通过同源重组修复(homologous recombination repair,HRR)、碱基错配修复(mismatch repair,MMR)和碱基切除修复(base excision repair,BER)等途径来修复的.作为真核生物研究的模式生物,对于酿酒酵母DNA损伤修复的HRR、MMR和BER途径研究颇多,也不断有一些新的成果出现,特别是对于相关途径的完善和相关蛋白的深化更是研究热点,在此对近年来有关重离子辐照酿酒酵母DNA损伤修复途径方面的研究做一综述.     

High mobility group (HMG) proteins: Modulators of chromatin structure and DNA repair in mammalian cells

It has been almost a decade since the last review appeared comparing and contrasting the influences that the different families of High Mobility Group proteins (HMGA, HMGB and HMGN) have on the various DNA repair pathways in mammalian cells. During that time considerable progress has been made in our understanding of how these non-histone proteins modulate the efficiency of DNA repair by all of the major cellular pathways: nucleotide excision repair, base excision repair, double-stand break repair and mismatch repair. Although there are often similar and over-lapping biological activities shared by all HMG proteins, members of each of the different families appear to have a somewhat ‘individualistic’ impact on various DNA repair pathways. This review will focus on what is currently known about the roles that different HMG proteins play in DNA repair processes and discuss possible future research areas in this rapidly evolving field.     

DNA repair BER pathway inhibition increases cell death caused by oxidative DNA damage in Trypanosoma cruzi

Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas disease, an endemic and neglected pathology in Latin America. It presents a life cycle that involves a hematophagous insect and man as well as domestic and wild mammals. The parasitic infection is not eliminated by the immune system of mammals; thus, the vertebrate host serves as a parasite reservoir. Additionally, chronic processes leading to dysfunction of the cardiac and digestive systems are observed. To establish a chronic infection some parasites should resist the oxidative damage to its DNA exerted by oxygen and nitrogen free radicals (ROS/RNS) generated in host cells. Till date there are no reports directly showing oxidative DNA damage and repair in T. cruzi. We establish that ROS/RNS generate nuclear and kinetoplastid DNA damage in T. cruzi that may be partially repaired by the parasite. Furthermore, we determined that both oxidative agents diminish T. cruzi cell viability. This effect is significantly augmented in parasites subsequently incubated with methoxyamine, a DNA base excision repair (BER) pathway inhibitor, strongly suggesting that the maintenance of T. cruzi viability is a consequence of DNA repair mechanisms.     

Discrimination and versatility in mismatch repair

Evolutionarily-conserved mismatch-repair (MMR) systems correct all or almost all base-mismatch errors from DNA replication via excision-resynthesis pathways, and respond to many different DNA lesions. Consideration of DNA polymerase error rates and possible consequences of excess gratuitous excision of perfectly paired (homoduplex) DNA in vivo suggests that MMR needs to discriminate against homoduplex DNA by three to six orders of magnitude. However, numerous binding studies using MMR base-mispair-recognition proteins, bacterial MutS or eukaryotic MSH2.MSH6 (MutSalpha), have typically shown discrimination factors between mismatched and homoduplex DNA to be 5-30, depending on the binding conditions, the particular mismatches, and the DNA-sequence contexts. Thus, downstream post-binding steps must increase MMR discrimination without interfering with the versatility needed to recognize a large variety of base-mismatches and lesions. We use a complex but highly MMR-active model system, human nuclear extracts mixed with plasmid substrates containing specific mismatches and defined nicks 0.15 kbp away, to measure the earliest quantifiable committed step in mismatch correction, initiation of mismatch-provoked 3'-5' excision at the nicks. We compared these results to binding of purified MutSalpha to synthetic oligoduplexes containing the same mismatches in the same sequence contexts, under conditions very similar to those prevailing in the nuclear extracts. Discrimination against homoduplex DNA, only two-to five-fold in the binding studies, increased to 60- to 230-fold or more for excision initiation, depending on the particular mismatches. Remarkably, the mismatch-preference order for excision initiation was substantially altered from the order for hMutSalpha binding. This suggests that post-binding steps not only strongly discriminate against homoduplex DNA, but do so by mechanisms not tightly constrained by initial binding preferences. Pairs of homoduplexes (40, 50, and 70 bp) prepared from synthetic oligomers or cut out of plasmids showed virtually identical hMutSalpha binding affinities, suggesting that high hMutSalpha binding to homoduplex DNA is not the result of misincorporations or lesions introduced during chemical synthesis. Intrinsic affinities of MutS homologs for perfectly paired DNA may help these proteins efficiently position themselves to carry out subsequent mismatch-specific steps in MMR pathways.     

NEIL2-initiated, APE-independent repair of oxidized bases in DNA: Evidence for a repair complex in human cells

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.     

Simulated microgravity decreases DNA repair capacity and induces DNA damage in human lymphocytes

The effect of simulated microgravity on DNA damage and apoptosis is still controversial. The objective of this study was to test whether simulated microgravity conditions affect the expression of genes for DNA repair and apoptosis. To achieve this objective, human lymphocyte cells were grown in a NASA‐developed rotating wall vessel (RWV) bioreactor that simulates microgravity. The same cell line was grown in parallel under normal gravitational conditions in culture flasks. The effect of microgravity on the expression of genes was measured by quantitative real‐time PCR while DNA damage was examined by comet assay. The result of this study revealed that exposure to simulated microgravity condition decreases the expression of DNA repair genes. Mismatch repair (MMR) class of DNA repair pathway were more susceptible to microgravity condition‐induced gene expression changes than base excision repair (BER) and nucleotide excision repair (NER) class of DNA repair genes. Downregulation of genes involved in cell proliferation (CyclinD1 and PCNA) and apoptosis (Bax) was also observed. Microgravity‐induced changes in the expression of some of these genes were further verified at the protein level by Western blot analysis. The findings of this study suggest that microgravity may induce alterations in the expression of these DNA repair genes resulting in accumulation of DNA damage. Reduced expression of cell‐cycle genes suggests that microgravity may cause a reduction in cell growth. Downregulation of pro‐apoptotic genes further suggests that extended exposure to microgravity may result in a reduction in the cells' ability to undergo apoptosis. Any resistance to apoptosis seen in cells with damaged DNA may eventually lead to malignant transformation of those cells. J. Cell. Biochem. 107: 723–731, 2009. © 2009 Wiley‐Liss, Inc.     

Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery

The p53 tumor suppressor that plays a central role in the cellular response to genotoxic stress was suggested to be associated with the DNA repair machinery which mostly involves nucleotide excision repair (NER). In the present study we show for the first time that p53 is also directly involved in base excision repair (BER). These experiments were performed with p53 temperature-sensitive (ts) mutants that were previously studied in in vivo experimental models. We report here that p53 ts mutants can also acquire wild-type activity under in vitro conditions. Using ts mutants of murine and human origin, it was observed that cell extracts overexpressing p53 exhibited an augmented BER activity measured in an in vitro assay. Depletion of p53 from the nuclear extracts abolished this enhanced activity. Together, this suggests that p53 is involved in more than one DNA repair pathway.     

Active DNA demethylation and DNA repair

DNA methylation on cytosine is an epigenetic modification and is essential for gene regulation and genome stability in vertebrates. Traditionally DNA methylation was considered as the most stable of all heritable epigenetic marks. However, it has become clear that DNA methylation is reversible by enzymatic “active” DNA demethylation, with examples in plant cells, animal development and immune cells. It emerges that “pruning” of methylated cytosines by active DNA demethylation is an important determinant for the DNA methylation signature of a cell. Work in plants and animals shows that demethylation occurs by base excision and nucleotide excision repair. Far from merely protecting genomic integrity from environmental insult, DNA repair is therefore at the heart of an epigenetic activation process.     

Spontaneous DNA repair in human mononuclear cells is calcium-dependent

Spontaneous DNA repair in peripheral blood mononuclear cells (PBMC) has been recently described. The aim of this study was to evaluate whether spontaneous DNA repair is Ca(2+)-dependent, as in vitro-stimulated DNA repair. Spontaneous DNA repair in PBMC was measured in a 1mM Ca2+ medium. The effect of extracellular Ca2+ chelation by EGTA, intracellular Ca2+ chelation by bapta-AM, and Ca2+ loading by the ionophore A23187 was examined. The signal transduction pathway was evaluated by inhibiting protein tyrosine kinase with genistein, calmodulin with W7, and calcineurin with cyclosporin A and tacrolimus. Extracellular Ca2+ chelation had no effect on spontaneous DNA repair, while both intracellular chelation and calcium overloading inhibited the DNA repair. Inhibition of protein tyrosine kinase, calmodulin or calcineurin reduced DNA repair. In conclusion, spontaneous DNA repair is mainly Ca(2+)-dependent at a narrow range of intracellular Ca2+ concentrations. The signal transduction cascade includes protein tyrosine kinase, calmodulin, and calcineurin.     

Role of excision mechanisms of DNA repair in induction of apoptosis

DNA repair and apoptosis lead to principally different final results: the first mechanism removes damages from DNA, restoring genome integrity; the second mechanism eliminates potentially dangerous cells harboring DNA lesions. The cells deficient in mismatch repair (MMR) demonstrate inceased resistance (viability) to DNA-damaging agents due to decreased ability to undergo apoptosis. This means that mechanism of MMR both restores structure of DNA and generates a signal for apoptosis. DNA breaks and single strand gaps, which are temporarily produced by excison mechanism during DNA repair, are suggested to be the initial signals for apoptosis. However pathway involved in such signaling at least partially is independent of p53 function.     

Ligation of newly replicated DNA controls the timing of DNA mismatch repair

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