Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Repair of alkylated bases in DNA is performed by O6-methylguanine-DNA methyltransferase (MGMT) and a set of enzymes of the base excision repair pathway involving N-methylpurine-DNA glycosylase (MPG), apurinic endonuclease (APE), DNA polymerase beta (Pol beta) and DNA ligase. The level of expression of these enzymes may exert a profound effect on resistance of cells towards alkylating drugs. We have comparatively analyzed the expression of MGMT and the different base excision repair genes in rat hepatoma cells (line H4IIE) after exposure to alkylating agents, X-rays and the glucocorticoid hormone dexamethasone. Furthermore, the effect of these agents on the activity of the cloned human MGMT promoter was assayed. Exposure of cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or ionizing radiation increased MGMT mRNA levels up to 4.5-fold. Under the same conditions of treatment, exerting only a weak toxic effect, MPG and DNA ligase I mRNA levels were not enhanced, whereas the amounts of APE and Pol beta mRNA transiently increased by approximately 2-fold after X-ray and MNNG treatment, respectively. Dexamethasone induced both MGMT, APE and Pol beta mRNA and the induction paralleled the increase in mRNA of the glucocorticoid-dependent gene tyrosine aminotransferase. The observed increase in MGMT mRNA was due to promoter activation, which was shown in transient transfection assays with MGMT promoter-CAT reporter constructs in H4IIE cells. In these assays, the human MGMT promoter was found to be induced by methylating agents (MNNG and methyl methanesulfonate), ionizing radiation and dexamethasone. Weak induction of the promoter was observed after UV irradiation. Treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate was ineffective in promoter activation. The transfected MGMT promoter was not inducible by mutagens in HeLa S3 cells, which do not respond with induction of the endogenous MGMT gene. This is the first report showing hormone induction of a DNA repair gene (MGMT). The induction of MGMT and other genes encoding enzymes involved in DNA alkylation damage repair may be relevant in cancer therapy by causing resistance of tumor cells to alkylating drugs.
 ...
PMID:Induction of the alkyltransferase (MGMT) gene by DNA damaging agents and the glucocorticoid dexamethasone and comparison with the response of base excision repair genes. 896 45

We examined expression of N-methylpurine-DNA glycosylase (MPG), a DNA repair enzyme that removes N-alkylpurine damage, in normal, malignant, and immortalized breast epithelial cells, and breast cancer cell lines (MDA-MB-231, MCF7, T47D). Northern analysis showed increased expression in cancer versus normal breast epithelial cells (2-24-fold). Southern blots revealed no gene amplification or polymorphisms. Immunofluorescence, immunohistochemistry, and Western blot analysis demonstrated increased MPG protein expression in the tumor cells that correlated with elevated glycosylase activity. Since MPG overexpression has been shown to be paradoxically associated with increased susceptibility to DNA damage, up-regulation of this gene may suggest a functional role in breast carcinogenesis.
 ...
PMID:Altered expression of the DNA repair protein, N-methylpurine-DNA glycosylase (MPG) in breast cancer. 968 56

Vinyl chloride is a known human and animal carcinogen that induces angiosarcomas of the liver. We review here studies on the formation and repair of DNA adducts associated with vinyl chloride and vinyl fluoride in exposed and control rodents and unexposed humans. These vinyl halides induce etheno (epsilon) adducts that are identical to those formed after lipid peroxidation. Of these adducts, N2,3-ethenoguanine (epsilon G) is present in greatest amounts in tissues of exposed animals. After exposure to vinyl chloride for four weeks, epsilon G levels attain steady-state concentrations, such that the amount of newly formed adducts equals the number of adducts that are lost each day. We report the first dosimetry of epsilon G in rats exposed to 0, 10, 100 or 1100 ppm vinyl chloride for five days or four weeks. The number of adducts increased in a supralinear manner. Exposure to 10 ppm vinyl chloride for five days caused a two- to threefold increase in epsilon G over that of the controls, while four weeks' exposure resulted in a fivefold increase. This was confirmed with [13C2]vinyl chloride and by measuring exogenous and endogenous adducts in the same animals. Exposure to 100 ppm vinyl chloride for four weeks caused a 25-fold increase in epsilon G levels over that found in control rats, while exposure to 1100 ppm resulted in a 42-fold increase. The amount of endogenous epsilon G was similar in liver DNA from rats and humans. A comparable response to exposure was seen in rats and mice exposed to 0, 25, 250 or 2500 ppm vinyl fluoride for 12 months. There was a very high correlation between epsilon G levels in rat and mouse liver at 12 months and the incidence of haemangiosarcoma at two years. We were able to demonstrate that the target cell population for angiosarcoma, the nonparenchymal cells, contained more epsilon G than hepatocytes, even though nonparenchymal cells are exposed by diffusion of vinyl halide metabolites formed in hepatocytes. The expression of N-methylpurine-DNA glycosylase mRNA was induced in rat liver after exposure to either 25 or 2500 ppm vinyl fluoride. When this induction was investigated in hepatocytes and nonparenchymal cells, it was found that the latter had only 20% of the N-methylpurine-DNA glycosylase mRNA of hepatocytes, and that only the hepatocytes had induction of this expression after exposure to vinyl fluoride. Thus, the target cells for vinyl halide carcinogenesis have much lower expression of this DNA repair enzyme, which has been associated with etheno adduct repair.
 ...
PMID:Formation and repair of DNA adducts in vinyl chloride- and vinyl fluoride-induced carcinogenesis. 1062 6

The mouse alkyladenine DNA glycosylase (Aag) initiates base excision repair with a broad substrate range that includes the highly mutagenic exocyclic etheno DNA base adduct 1,N6-ethenodeoxyadenosine ((epsilon)dA). Previous attempts to determine the in vivo role of Aag in (epsilon)dA repair were complicated by technological difficulties in measuring low levels of (epsilon)dA in genomic DNA. Here we describe the development of a new method for (epsilon)dA detection in genomic DNA that couples an immunoaffinity purification with LC-MS/MS analysis and that utilizes an isotopically labeled internal standard. We go on to describe the application of this method to measuring the in vivo repair of (epsilon)dA base lesions in liver and lung tissue of wild type and Aag null mice. Our results demonstrate that while Aag clearly represents the major DNA repair enzyme for the in vivo removal (epsilon)dA bases, these lesions can also be eliminated from the genome via an alternative mechanism.
 ...
PMID:New immunoaffinity-LC-MS/MS methodology reveals that Aag null mice are deficient in their ability to clear 1,N6-etheno-deoxyadenosine DNA lesions from lung and liver in vivo. 1517 41

Mutagenesis of protein-encoding sequences occurs ubiquitously; it enables evolution, accumulates during aging, and is associated with disease. Many biotechnological methods exploit random mutations to evolve novel proteins. To quantitate protein tolerance to random change, it is vital to understand the probability that a random amino acid replacement will lead to a protein's functional inactivation. We define this probability as the "x factor." Here, we develop a broadly applicable approach to calculate x factors and demonstrate this method using the human DNA repair enzyme 3-methyladenine DNA glycosylase (AAG). Three gene-wide mutagenesis libraries were created, each with 10(5) diversity and averaging 2.2, 4.6, and 6.2 random amino acid changes per mutant. After determining the percentage of functional mutants in each library using high-stringency selection (>19,000-fold), the x factor was found to be 34% +/- 6%. Remarkably, reanalysis of data from studies of diverse proteins reveals similar inactivation probabilities. To delineate the nature of tolerated amino acid substitutions, we sequenced 244 surviving AAG mutants. The 920 tolerated substitutions were characterized by substitutability index and mapped onto the AAG primary, secondary, and known tertiary structures. Evolutionarily conserved residues show low substitutability indices. In AAG, beta strands are on average less substitutable than alpha helices; and surface loops that are not involved in DNA binding are the most substitutable. Our results are relevant to such diverse topics as applied molecular evolution, the rate of introduction of deleterious alleles into genomes in evolutionary history, and organisms' tolerance of mutational burden.
 ...
PMID:Protein tolerance to random amino acid change. 1519 60

The base excision repair pathway removes damaged DNA bases and resynthesizes DNA to replace the damage. Human alkyladenine DNA glycosylase (AAG) is one of several damage-specific DNA glycosylases that recognizes and excises damaged DNA bases. AAG removes primarily damaged adenine residues. Human AP endonuclease 1 (APE1) recognizes AP sites produced by DNA glycosylases and incises the phophodiester bond 5' to the damaged site. The repair process is completed by a DNA polymerase and DNA ligase. If not tightly coordinated, base excision repair could generate intermediates that are more deleterious to the cell than the initial DNA damage. The kinetics of AAG-catalyzed excision of two damaged bases, hypoxanthine and 1,N6-ethenoadenine, were measured in the presence and absence of APE1 to investigate the mechanism by which the base excision activity of AAG is coordinated with the AP incision activity of APE1. 1,N6-ethenoadenine is excised significantly slower than hypoxanthine and the rate of excision is not affected by APE1. The excision of hypoxanthine is inhibited to a small degree by accumulated product, and APE1 stimulates multiple turnovers by alleviating product inhibition. These results show that APE1 does not significantly affect the kinetics of base excision by AAG. It is likely that slow excision by AAG limits the rate of AP site formation in vivo such that AP sites are not created faster than can be processed by APE1.
 ...
PMID:Slow base excision by human alkyladenine DNA glycosylase limits the rate of formation of AP sites and AP endonuclease 1 does not stimulate base excision. 1701 65

Glioblastoma Multiforme (GBM), the most common and lethal primary human brain tumor, exhibits multiple molecular aberrations. We report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumor suppressor gene (TSG). Although GATA4 was expressed in normal brain, loss of GATA4 was observed in 94/163 GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor-like phenotype, formation of neurospheres, and the ability to differentiate into astrocytes, neurons, and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures, and brain tumor-initiating cells suppressed tumor growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21(CIP1), independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT (O-6-methylguanine-DNA-methyltransferase), the DNA repair enzyme which is often implicated in temozolomide resistance. Instead, GATA4 reduced expression of APNG (alkylpurine-DNA-N-glycosylase), a DNA repair enzyme which is poorly characterized in GBM-mediated temozolomide resistance. Identification and validation of GATA4 as a TSG and its downstream targets in GBM may yield promising novel therapeutic strategies.
 ...
PMID:A GATA4-regulated tumor suppressor network represses formation of malignant human astrocytomas. 2146 20

During its life cycle, the protist parasite Entamoeba histolytica encounters reactive oxygen and nitrogen species that alter its genome. Base excision repair (BER) is one of the most important pathways for the repair of DNA base lesions. Analysis of the E. histolytica genome revealed the presence of most of the BER components. Surprisingly, this included a gene encoding an apurinic/apyrimidinic (AP) endonuclease that previous studies had assumed was absent. Indeed, our analysis showed that the genome of E. histolytica harbors the necessary genes needed for both short and long-patch BER sub-pathways. These genes include DNA polymerases with predicted 5'-dRP lyase and strand-displacement activities and a sole DNA ligase. A distinct feature of the E. histolytica genome is the lack of several key damage-specific BER glycosylases, such as OGG1/MutM, MDB4, Mag1, MPG, SMUG, and TDG. Our evolutionary analysis indicates that several E. histolytica DNA glycosylases were acquired by lateral gene transfer (LGT). The genes that encode for MutY, AlkD, and UDG (Family VI) are included among these cases. Endonuclease III and UNG (family I) are the only DNA glycosylases with a eukaryotic origin in E. histolytica. A gene encoding a MutT 8-oxodGTPase was also identified that was acquired by LGT. The mixed composition of BER genes as a DNA metabolic pathway shaped by LGT in E. histolytica indicates that LGT plays a major role in the evolution of this eukaryote. Sequence and structural prediction of E. histolytica DNA glycosylases, as well as MutT, suggest that the E. histolytica DNA repair proteins evolved to harbor structural modifications that may confer unique biochemical features needed for the biology of this parasite.
 ...
PMID:Evolution of Base Excision Repair in Entamoeba histolytica is shaped by gene loss, gene duplication, and lateral gene transfer. 3082 89

The DNA repair enzyme AAG has been shown in mice to promote tissue necrosis in response to ischaemic reperfusion or treatment with alkylating agents. A chemical probe inhibitor is required for investigations of the biological mechanism causing this phenomenon and as a lead for drugs that are potentially protective against tissue damage from organ failure and transplantation, and alkylative chemotherapy. Herein, we describe the rationale behind the choice of arylmethylpyrrolidines as appropriate aza-nucleoside mimics for an inhibitor followed by their synthesis and the first use of a microplate-based assay for quantification of their inhibition of AAG. We finally report the discovery of an imidazol-4-ylmethylpyrrolidine as a fragment-sized, weak inhibitor of AAG.
 ...
PMID:An aza-nucleoside, fragment-like inhibitor of the DNA repair enzyme alkyladenine glycosylase (AAG). 3232 52