Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mRNA for the epithelial Na(+) channel gamma subunit (gammaENaC) is regulated developmentally in the lung, colon and distal nephron and in response to Na(+) deprivation and systemic corticosteroids in the distal colon. Because such regulation is likely to be at the level of gene transcription, we examined the function of the promoter and other 5' flanking elements of the human gammaENaC gene. The proximal 5' flanking region contains two GC boxes but does not contain a TATA box. A 450 bp human gammaENaC fragment (-459 to +40) directed the expression of luciferase in H441 cells and primer extension analysis in transfected cells confirmed the correct initiation of human gammaENaC-luciferase chimaeric transcripts. By deletional analysis, GC boxes at -21 and -52 were found to be critical for this promoter activity. To begin to identify transcription factors that bind to the core promoter, a double-stranded oligonucleotide that corresponded to this region was synthesized and tested in a gel mobility-shift assay. Incubation of this radiolabelled oligonucleotide with nuclear extracts from H441 and FRTL5 cells resulted in the formation of four specific and distinct DNA-protein complexes. On the basis of antibody 'supershift' assays, one of these factors corresponds to Sp1, whereas the other three correspond to Sp3. Further upstream, an approx. 300 nt (-1143 to -839) polypurine-polypyrimidine tract (PPy tract) containing internal mirror repeats was identified. When contained in a supercoiled plasmid, the approx. 1200 nt 5' flanking region was sensitive to S1 endonuclease, which was consistent with the formation of an intramolecular triplex DNA ('H-DNA') structure with an unpaired single strand. High-resolution mapping with S1 endonuclease and sequencing of S1-generated clones confirmed that all S1-sensitive sites were within the PPy tract. Finally, a negative regulatory element was identified between -1525 and -1296 that functioned in lung, colon and collecting duct cell lines.
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PMID:Human amiloride-sensitive epithelial Na+ channel gamma subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for triplex DNA formation. 1072 8

Abasic (apurinic/apyrimidinic or AP) sites are a frequent type of DNA damage that threatens genetic stability. The predominant mammalian enzyme initiating repair of AP sites is the Ape1 AP endonuclease (also called Apex or Hap1), which also facilitates DNA binding by several transcription factors (Ref1 activity). We found that expression of the APE1 gene was coordinated with the cell cycle in murine NIH3T3 cells: APE1 mRNA levels rose after the G(1)-S transition and peaked approximately 4-fold higher in early to mid-S phase. The increased APE1 mRNA was the result of transcriptional activation rather than increased mRNA stability. Fusions of various APE1 promoter fragments to the chloramphenicol acetyltransferase CAT reporter gene indicated that APE1 expression depends on two transcription factor Sp1 binding sites within the promoter region. Mutation of these sites or of two CCAAT elements within the APE1 promoter, in conjunction with protein binding studies, demonstrated their specific roles. The Sp1 site upstream of the transcription start, together with an adjacent CCAAT element, establishes a protein-DNA complex required for basal transcription of APE1. The Sp1 site downstream of the transcription start was required for the response to cell growth. Because Ape1 is a dual function enzyme, its cell cycle-dependent expression might affect both DNA repair and the activity of various transcription factors as a function of the cell cycle.
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PMID:Key role of a downstream specificity protein 1 site in cell cycle-regulated transcription of the AP endonuclease gene APE1/APEX in NIH3T3 cells. 1155 53

Twin and family studies have demonstrated that a large part of a population's variance in bone mineral density (BMD) is attributable to genetic factors. A polymorphism in the collagen type I alpha1 (COLIA1) gene has recently been associated with low bone mass and fracture incidence. We analyzed the relationship between COLIA1 gene polymorphism, lumbar spine and hip BMD, and fracture prevalence in a population of 319 postmenopausal women classified by WHO standards, including 98 nonosteoporotic women (NOPW) and 221 osteoporotic postmenopausal women (OPW), divided into 139 osteoporotic women without fracture (OPWnF) and 82 osteoporotic women with fracture (OPWwF). The COLIA1 genotype was assessed by polymerase chain reaction and BalI endonuclease digestion. Genotype frequencies for the total group were 49.2% GG homozygotes, 39.5% GT heterozygotes, and 11.3% TT homozygotes. We found significant differences in the percentage of homozygous TT between NOPW and OPW (6.1% and 13.6%, respectively). Significantly, the occurrence of genotype TT in OPWnF was 6.2%, and 28% in OPWwF. We observed no associations between the COLIA1 genotype and lumbar spine and hip BMD. The prevalence of fractures varied significantly by genotype: GG, 26.1%; GT, 15.9%; and TT, 58.3%. Logistic regression analysis of fracture prevalence showed that, for prevalent fractures, the women with the TT genotype had a 5.9-fold increased risk when compared with the other genotypes (GG + GT). When prevalence was adjusted for age, body mass index, and BMD, the fracture risk was 4.8 for the TT group vs. the genotype GG, whereas it was 0.6 for the GT genotype. In conclusion, we found the COLIA1 Sp1 TT genotype to be associated with an increased fracture risk in postmenopausal women. Interestingly, this genotype-dependent risk could not be explained completely by BMD differences.
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PMID:Polymorphism in the type I collagen (COLIA1) gene and risk of fractures in postmenopausal women. 1179 89

HEX1/hExo1 is a Class III nuclease of the RAD2 family with 5' to 3' exonuclease and flap structure-specific endonuclease activities. HEX1/hExo1 is expressed at low levels in a wide variety of tissues, but at higher levels in fetal liver and adult bone marrow, suggesting HEX1/hExo1 is important for hematopoietic stem cell development. A putative HEX1/hExo1 promoter fragment extending from -6240 to +1600bp exhibits cell-type specific activity in transient transfection assays. This fragment directs high luciferase reporter gene expression in the hematopoietic cell line K562, chronic myelogenous leukemia cells, but low luciferase expression in the non-hematopoietic cell line HeLa, human cervical carcinoma cells. Deletion studies identified a fragment spanning -688 to +1600bp that exhibits full transcriptional activity while a slightly shorter fragment from -658 to +1600bp exhibits significantly decreased promoter activity. In vitro binding assays revealed DNA-binding activities that interact with -687 to -681bp and -665 to -658bp elements. Oligonucleotide competition and antibody disruption studies determined that the transcription factor CREB-1 recognizes the -687 to -681bp element, while transcription factors Sp1 and Sp3 recognize the -665 to -658bp element. Mutation of either the CREB-1 or Sp1/Sp3 binding sites dramatically reduces HEX1/hExo1 promoter activity and elimination of both elements abolishes promoter activity.
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PMID:Identification of the human HEX1/hExo1 gene promoter and characterization of elements responsible for promoter activity. 1253 89

Single-stranded DNA genomes have been constructed that site-specifically contain the 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxoG) oxidation products guanidinohydantoin (Gh) and the two stable stereoisomers of spiroiminodihydantoin (Sp1 and Sp2). The circular viral genomes were transfected into wild-type AB1157 Escherichia coli, and the efficiency of lesion bypass by DNA polymerase(s) was assessed. Viral progeny were analyzed for mutation frequency and type using the recently developed restriction endonuclease and postlabeling (REAP) assay. Gh was bypassed nearly as efficiently as the parent 8-oxoG but was highly mutagenic, causing almost exclusive G --> C transversions. The stereoisomers Sp1 and Sp2 were, in comparison, much stronger blocks to DNA polymerase extension and caused a mixture of G --> T and G --> C transversions. The ratio of G --> T to G --> C mutations for each Sp lesion was dependent on the stereochemical configuration of the base. All observed mutation frequencies were at least an order of magnitude higher than those caused by 8-oxoG. Were these lesions to be formed in vivo, our data show that they are absolutely miscoding and may be refractory to repair after translesion synthesis.
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PMID:The hydantoin lesions formed from oxidation of 7,8-dihydro-8-oxoguanine are potent sources of replication errors in vivo. 1289 11

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53(+/+), but not in the isogenic p53 null mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 null cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.
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PMID:Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage. 1820 37

We previously described a population of regulatory macrophages that produced high levels of IL-10 and low levels of IL-12/23. We now describe and characterize the expression of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) by these macrophages. HB-EGF has previously been associated with a number of physiological and pathological conditions, including tumor growth and angiogenesis. The induction of HB-EGF in regulatory macrophages is due to new transcription and not to increased mRNA stability. The transcription factor Sp1 is a major factor in HB-EGF production, and knockdown of Sp1 substantially diminishes HB-EGF production. Sp1 was recruited to three sites within the first 2 kb of the HB-EGF promoter following stimulation, and the site located at -83/-54 was required for HB-EGF promoter activity. These regions of the promoter become more accessible to endonuclease activity following macrophage activation, and this accessibility was contingent on activation of the MAPK, ERK. We show that several experimental manipulations that give rise to regulatory macrophages also result in HB-EGF production. These observations indicate that in addition to the secretion of the anti-inflammatory cytokine IL-10, another novel characteristic of regulatory macrophages is the production of angiogenic HB-EGF.
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PMID:The expression of heparin-binding epidermal growth factor-like growth factor by regulatory macrophages. 1920 46

DNA constantly undergoes chemical modification due to endogenous and exogenous mutagens. The DNA base excision repair (BER) pathway is the frontline mechanism handling the majority of these lesions, and primarily involves a DNA incision and subsequent resealing step. It is imperative that these processes are extremely well-coordinated as unrepaired DNA single strand breaks (SSBs) can be converted to DNA double strand breaks during replication thus triggering genomic instability. However, the mechanism(s) governing the BER process are poorly understood. Here we show that accumulation of unrepaired SSBs triggers a p53/Sp1-dependent downregulation of APE1, the endonuclease responsible for the DNA incision during BER. Importantly, we demonstrate that impaired p53 function, a characteristic of many cancers, leads to a failure of the BER coordination mechanism, overexpression of APE1, accumulation of DNA strand breaks and results in genomic instability. Our data provide evidence for a previously unrecognized mechanism for coordination of BER by p53, and its dysfunction in p53-inactivated cells.
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PMID:p53 coordinates base excision repair to prevent genomic instability. 2677 55


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