Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue (Cys-252) in the DNA binding domain. Jun protein inactivated by oxidation of this residue can be efficiently reactivated by a factor from human cell nuclei, recently identified as a DNA repair enzyme (termed HAP1 or Ref-1). The HAP1 protein consists of a core domain, which is highly conserved in a family of prokaryotic and eukaryotic DNA repair enzymes, and a 61-amino-acid N-terminal domain absent from bacterial homologs such as Escherichia coli exonuclease III. The eukaryote-specific N-terminal domain was dispensable for the DNA repair functions of the HAP1 protein but was essential for reactivation of the DNA binding activity of oxidized Jun protein. Consistent with this finding, exonuclease III protein could not reactive Jun. A minimal 26-residue region of the N-terminal domain proximal to the core of the HAP1 enzyme was required for redox activity. By site-directed mutagenesis, cysteine 65 was identified as the redox active site in the HAP1 enzyme. In addition, it is proposed that cysteine 93 interacts with the redox active site, probably via disulfide bridge formation. It is concluded that the HAP1 protein has evolved a novel redox activation domain capable of regulating the DNA binding activity of a proto-oncogene product which is not essential for its DNA repair functions. Identification of a putative active site cysteine residue should facilitate analysis of the mechanism by which the HAP1 protein may alter the redox state of a wide range of transcription factors.
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PMID:Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding. 835 88

APEX nuclease is a mammalian DNA repair enzyme having apurinic/apyrimidinic (AP) endonuclease, 3'-5'-exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities. It is also a redox factor (Ref-1), stimulating DNA binding activity of AP-1 binding proteins such as Fos and Jun. In the present paper, a cDNA for the enzyme was isolated from a rat brain cDNA library using mouse Apex cDNA as a probe and sequenced. The rat Apex cDNA was 1221 nucleotides (nt) long, with a 951-nt coding region. The amino acid sequence of rat APEX nuclease has 98.4% identity with mouse APEX nuclease. Using the rat Apex cDNA as a probe for Northern blot analysis, the size of rat Apex mRNA was shown to be approximately 1.5 kb. Its expression was compared in 9 rat organs on postnatal days 7 and 28. Although Apex mRNA was expressed ubiquitously, the levels varied significantly, suggesting organ- or tissue-specific expression of the Apex gene. The highest level was observed in the testis, relatively high levels in the thymus, spleen, kidney and brain, and the lowest level in the liver. The level of expression at postnatal day 28, with the exception of the testis, was almost the same as or lower in respective organs than that at postnatal day 7. Postnatal developmental changes of Apex mRNA expression in the testis and thymus were further studied. The expression in testis was markedly increased on postnatal days 21 and 28. The expression in thymus increased once at postnatal day 14, and then decreased. The developmental changes of Apex mRNA expression in testis and thymus suggest that APEX nuclease is involved in processes such as recombinational events.
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PMID:cDNA cloning of rat major AP endonuclease (APEX nuclease) and analyses of its mRNA expression in rat tissues. 870 82

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.
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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

The human DNA repair enzyme apurinic/apyrimidinic endonuclease (APE/ref-1) is a multifunctional protein in the DNA base excision repair (BER) pathway that is responsible for repair of apurinic/apyrimidinic (AP) sites in DNA. DNA repair and programmed cell death both function using different mechanisms to protect the organism from the consequences of extensive cellular damage; however, little is known about the relationship of the DNA BER repair pathway to apoptosis. We have determined the relationship of a BER DNA repair enzyme, APE, to apoptosis using the myeloid leukemia cell line HL-60, which can be induced to differentiate down the granulocytic or monocytic/ macrophage pathway. Treatment of HL-60 cells with retinoic acid/DMSO (granulocytic) or phorbol 12-myristate 13-acetate (monocytic) results in apoptosis and in down-regulation of APE expression at both the RNA and protein levels. Moreover, double-labeling experiments using APE immunohistochemistry and the terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling assay for apoptosis demonstrate that individual cells undergoing apoptosis lose expression of APE regardless of their state of differentiation. Blocking apoptosis by overexpression of the bcl-2 proto-oncogene in HL-60 cells or by a bcr-abl-related mechanism in K562 cells and subsequent differentiation results in morphological differentiation but no loss of APE expression. These studies establish that down-regulation of APE expression is associated with programmed cell death in cells of the myeloid lineage.
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PMID:Down-regulation of apurinic/apyrimidinic endonuclease expression is associated with the induction of apoptosis in differentiating myeloid leukemia cells. 910 Oct 90

Thioredoxin (TRX) is a pleiotropic cellular factor that has thiol-mediated redox activity and is important in regulation of cellular processes, including proliferation, apoptosis, and gene expression. The activity of several transcription factors is posttranslationally altered by redox modification(s) of specific cysteine residue(s). One such factor is nuclear factor (NF)-kappa B, whose DNA-binding activity is markedly augmented by TRX treatment in vitro. Similarly, the DNA-binding activity of activator protein 1 (AP-1) is modified by a DNA repair enzyme, redox factor 1 (Ref-1), which is identical to a DNA repair enzyme, AP endonuclease. Ref-1 activity is in turn modulated by various redox-active compounds, including TRX. We here report the molecular cascade of redox regulation of AP-1 mediated by TRX and Ref-1. Phorbol 12-myristate 13 acetate efficiently translocated TRX into the HeLa cell nucleus where Ref-1 preexists. This process seems to be essential for AP-1 activation by redox modification because co-overexpression of TRX and Ref-1 in COS-7 cells potentiated AP-1 activity only after TRX was transported into the nucleus by phorbol 12-myristate 13 acetate treatment. To prove the direct active site-mediated association between TRX and Ref-1, we generated a series of substitution-mutant cysteine residues of TRX. In both an in vitro diamide-induced cross-linking study and an in vivo mammalian two-hybrid assay we proved that TRX can associate directly with Ref-1 in the nucleus; also, we demonstrated the requirement of cysteine residues in the TRX catalytic center for the potentiation of AP-1 activity. This report presents an example of a cascade in cellular redox regulation.
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PMID:AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. 910 29

Mitochondria have been proposed to possess base excision repair processes to correct oxidative damage to the mitochondrial genome. As the only DNA polymerase (pol) present in mitochondria, pol gamma is necessarily implicated in such processes. Therefore, we tested the ability of the catalytic subunit of human pol gamma to participate in uracil-provoked base excision repair reconstituted in vitro with purified components. Subsequent to actions of uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease, human pol gamma was able to fill a single nucleotide gap in the presence of a 5' terminal deoxyribose phosphate (dRP) flap. We report here that the catalytic subunit of human pol gamma catalyzes release of the dRP residue from incised apurinic/apyrimidinic sites to produce a substrate for DNA ligase. The heat sensitivity of this activity suggests the dRP lyase function requires a three-dimensional protein structure. The dRP lyase activity does not require divalent metal ions, and the ability to trap covalent enzyme-DNA complexes with NaBH4 strongly implicates a Schiff base intermediate in a beta-elimination reaction mechanism.
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PMID:Identification of 5'-deoxyribose phosphate lyase activity in human DNA polymerase gamma and its role in mitochondrial base excision repair in vitro. 977 Apr 71

A study was made of the relationship between the intrinsic radiosensitivity of human cervical tumours and the expression of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (HAP1). The radiosensitivity of clonogenic cells in tumour biopsies was measured as surviving fraction at 2 Gy (SF2) using a soft agar assay. HAP1 expression levels were determined after staining of formalin-fixed paraffin-embedded tumour sections with a rabbit antiserum raised against recombinant HAP1. Both measurements were obtained on pretreatment biopsy material. All 25 tumours examined showed positive staining for HAP1, but there was heterogeneity in the level of expression both within and between tumours. The average coefficients of variation for intra- and intertumour heterogeneity were 62% and 82% respectively. There was a moderate but significant positive correlation between the levels of HAP1 expression and SF2 (r = 0.60, P = 0.002). Hence, this study shows that there is some relationship between intrinsic radiosensitivity and expression of a DNA repair enzyme in cervical carcinomas. The results suggest that this type of approach may be useful in the development of rapid predictive tests of tumour radiosensitivity.
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PMID:Levels of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (APE1, APEX, Ref-1) are associated with the intrinsic radiosensitivity of cervical cancers. 982 Jan 67

Chronic oxidative stress has been hypothesized to be a major contributor to the aging process. The continued exposure to reactive oxygen species (ROS) generated by oxidative metabolism or environmental sources can damage critical cellular structures and be responsible for some age-related pathology. The exposure of rodents to 100% oxygen, isobaric hyperoxia, increases ambient ROS levels and significantly increases apoptosis in brain. The deleterious effects of ROS also include increased lipid peroxidation, protein oxidation, and DNA damage. Although differences in the relative amounts of oxidative stress in young and old brains have been observed, the mechanisms responsible for impaired aging-associated DNA repair processes have not been characterized. We measured DNA levels of the DNA repair enzyme apurinic/apyrimidinic endonuclease (APE/Ref-1) protein by Western blot analysis in the brains of young (3-month) and old (30-month) male rats exposed to isobaric hyperoxia. Given that APE/Ref-1 is the rate-limiting enzyme in the repair pathway of apurinic/apyrimidinic sites generated in DNA by oxidative damage, we assumed that APE/Ref-1 protein levels were a good reflection of ongoing DNA base excision repair. Isobaric hyperoxia stimulated APE/Ref-1 expression in the hippocampus and basal forebrain of young rats experiencing 100% oxygen for 6 hr, while aged rats showed no significant changes in APE/Ref-1 protein levels in all brain areas at any time tested (0-48 hr) after hyperoxia. Differences in the stress-induced levels of expression of DNA repair enzymes may contribute to apoptotic increases and pathology associated with the aging process.
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PMID:APE/Ref-1 responses to oxidative stress in aged rats. 984 54

Cerebral ischemia and the aftermath of reperfusion form a hypoxic/hyperoxic sequence of events that can trigger oxidative stress response cascades in neurons of the central nervous system. After transient ischemia there is an increase in intracellular Ca2+ release, extracellular glutamate, reactive oxygen species (ROS) and nitric oxide, genotoxic events that stimulate DNA repair. Increased oxidative stress and interrupted blood flow in ischemia, like DNA repair, also deplete cellular ATP and commit neurons to apoptosis. We report that levels of the DNA repair enzyme apurinic/apyrimidinic endonuclease (APE/Ref-1) decreased significantly in the hippocampus but not other brain areas after 6 h of reperfusion following an induced ischemic insult. This specific inhibition of APE/Ref-1 expression may affect the extent of apoptosis after ischemia.
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PMID:APE/Ref-1 responses to ischemia in rat brain. 992 39

Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.
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PMID:Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate. 1032 34


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