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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The tumor suppressor genes BRCA1 and BRCA2, which confer increased susceptibility to breast and (or) ovarian cancer, were recently identified. Mutation analysis of BRCA1 has demonstrated significant allelic heterogeneity; however, some distinct mutations have been detected in unrelated individuals. The most notable is the 185delAG mutation, which occurs at an estimated frequency of approximately 1% in individuals of Ashkenazi Jewish descent [1]. Although consensus has not been reached regarding clinical testing for mutations in BRCA1, a tiered strategy may be appropriate, in which direct testing for the more common mutations is one component. Specific alleles can be detected by using PCR-mediated site-directed mutagenesis (PSM), which alters the PCR products derived from either the wild-type or mutant allele to create or destroy a restriction endonuclease recognition site. Recognition sites are introduced by a base substitution in one of the primers. The alleles are then resolved by electrophoresis of the digested PCR products. We have applied this technique to the detection of four BRCA1 mutations: 185delAG, 5382insC, E1250X, and R1443X. Another mutation, 1294de140, can be resolved from the wild-type allele by high-resolution gel electrophoresis alone. The PSM technique is sensitive, does not require radioactivity, and is specific for individual mutations.
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PMID:Direct detection of mutations in the breast and ovarian cancer susceptibility gene BRCA1 by PCR-mediated site-directed mutagenesis. 899 Feb 12

We introduce a novel experimental strategy for DNA mutation detection named the Mismatch Identification DNA Analysis System (MIDAS) [1, 2], which has an associated isothermal probe amplification step to increase target DNA detection sensitivity to attomole levels. MIDAS exploits DNA glycosylases to remove the sugar moiety on one strand (the probe strand) at a DNA base pair mismatch. The resulting apyrimidinic/ apurinic (AP) site is cleaved by AP endonucleases/lyases either associated with the DNA glycosylase or externally added to the reaction mixture. MIDAS utilizes 32p- or FITC-labeled oligonucleotides as mutation probes. Generally between 20-50 nucleotides in length, the probe hybridizes to the target sequence at the reaction temperature. Mismatch repair enzymes (MREs) then cut the probe at the point of mismatch. Once the probe is cleaved, the fragments become thermally unstable and fall off the target, thereby allowing another full-length probe to hybridize. This oscillating process amplifies the signal (cleaved probe). Cleavage products can be detected by electrophoretic separation followed by autoradiography, or by laser-induced fluorescence-capillary electrophoresis (LIF-CE) of fluorophore-labeled probes in two minutes using a novel CE matrix. In the present experiments, we employed the mesophilic Escherichia coli enzyme deoxyinosine 3'-endonuclease (Endo V), and a novel thermostable T/G DNA glycosylase, TDG mismatch repair enzyme (TDG-MRE). MIDAS differentiated between a clinical sample BRCA 1 wild-type sequence and a BRCA1 185delAG mutation without the need for polymerase chain reaction (PCR). The combination of MIDAS with LIF-CE should make detection of known point mutations, deletions, and insertions a rapid and cost-effective technique well suited for automation.
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PMID:Mutation identification DNA analysis system (MIDAS) for detection of known mutations. 1038 Jul 53

A novel approach to mutation screening in the large exon 11 (comprising 3427 bp) of the human BRCA1 gene is presented. Restriction endonuclease fingerprinting single-strand conformation polymorphism (REF-SSCP) is based on repeated detection of DNA sequence variants in different restriction endonuclease fragments, and we evaluated the method using blood samples from 25 Norwegian patients with hereditary breast/ovarian cancer. We compared REF-SSCP to constant denaturant gel electrophoresis (CDGE) and to the protein truncation test (PTT). REF-SSCP detected 12 different DNA variants. Four of these were not detected by CDGE, and only one variant detected by CDGE was missed by REF-SSCP. PTT detected 4 of these 13 variants. REF-SSCP was subsequently applied to a second patient series (Swedish, n=20). A total of 14 different DNA variants were detected by REF-SSCP, 6 of which were truncating mutations (PTT detected only 4). Nonsense and frameshift mutations that are putative breast/ovarian cancer mutations, were detected in 7 of the 25 Norwegian and 9 of the 20 Swedish patients.
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PMID:Enhanced detection of mutations in BRCA1 exon 11 using restriction endonuclease fingerprinting-single-strand conformation polymorphism. 1119 32

Efficient genetic analysis of large exonic regions containing heterozygous mutations and common polymorphisms can be difficult. We have analyzed 30 patients for inherited susceptibility mutations (ISM) within exon 11 of the BRCA1 gene as part of an ongoing genetic epidemiological study of high-risk breast cancer (HRBC). A novel combination of restriction endonuclease fingerprinting (REF) and conformation sensitive gel electrophoresis (CSGE) was developed for rapid and efficient screening of mutations. This method (REF-CSGE) was compared side-by-side with standard CSGE and evaluated for both efficiency and sensitivity of detection. REF-CSGE detected 100% of the alterations found by CSGE. However, one variant was only detectable by REF-CSGE. All samples with variant bands were sequenced to confirm the nature of the alteration. In total, two small deletions (frameshifts) and 62 point mutations (60 known polymorphisms and two variants of unknown significance) were found in our cohort. The majority of the exon 11 polymorphisms detected are inherited as a linked haplotype. Point mutations that comprise these haplotypes could be simultaneously detected on a single gel by REF-CSGE, thereby decreasing the number of sequencing reactions necessary to elucidate heteroduplex patterns seen on CSGE gels. An analysis of the overall efficiency of both techniques revealed that REF-CSGE required 67% fewer confirmatory sequencing reactions, resulting in savings in both reagents and technician time.
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PMID:Restriction endonuclease fingerprinting enhanced conformation sensitive gel electrophoresis (REF-CSGE) in the analysis of BRCA1 exon 11 mutations in a high-risk breast cancer cohort. 1200 22

BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with the Rad50.Mre11.Nbs1 complex, which occupies a central role in DNA double strand break repair mediated by nonhomologous end joining (NHEJ) and homologous recombination. BRCA1 has been shown to be required for homology-directed recombination repair. However, the role of BRCA1 in NHEJ, a critical pathway for the repair of double strand breaks and genome stability in mammalian cells, remains elusive. Here, we established a pair of mouse embryonic fibroblasts (MEFs) derived from 9.5-day-old embryos with genotypes Brca1(+/+):p53(-/-) or Brca1(-/-):p53(-/-). The Brca1(-/-):p53(-/-) MEFs appear to be extremely sensitive to ionizing radiation. The contribution of BRCA1 in NHEJ was evaluated in these cells using three different assay systems. First, transfection of a linearized plasmid in which expression of the reporter gene required precise end joining indicated that Brca1(-/-) MEFs display a moderate deficiency when compared with Brca1(+/+) cells. Second, using a retrovirus infection assay dependent on NHEJ, a 5-10-fold reduction in retroviral integration efficiency was observed in Brca1(-/-) MEFs when compared with the Brca1(+/+) MEFs. Third, Brca1(-/-) MEFs exhibited a 50-100-fold deficiency in microhomology-mediated end-joining activity of a defined chromosomal DNA double strand break introduced by a rare cutting endonuclease I-SceI. These results provide evidence that Brca1 has an essential role in microhomology-mediated end joining and suggest a novel molecular basis for its caretaker role in the maintenance of genome integrity.
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PMID:BRCA1 facilitates microhomology-mediated end joining of DNA double strand breaks. 1203 51

Previous studies have shown that expansion-prone repeats form structures that inhibit human flap endonuclease (FEN-1). We report here that faulty processing by FEN-1 initiates repeat instability in mammalian cells. Disease-length CAG tracts in Huntington's disease mice heterozygous for FEN-1 display a tendency toward expansions over contractions during intergenerational inheritance compared to those in homozygous wild-type mice. Further, with regard to human cells expressing a nuclease-defective FEN-1, we provide direct evidence that an unprocessed FEN-1 substrate is a precursor to instability. In cells with no endogenous defects in DNA repair, exogenous nuclease-defective FEN-1 causes repeat instability and aberrant DNA repair. Inefficient flap processing blocks the formation of Rad51/BRCA1 complexes but invokes repair by other pathways.
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PMID:Nuclease-deficient FEN-1 blocks Rad51/BRCA1-mediated repair and causes trinucleotide repeat instability. 1291 30

Incubation of gradient purified human spermatozoa, which are routinely maintained in media prior to IVF and intracytoplasmic sperm injection (ICSI), induced DNA strand breaks (up to 89 nicks x 10(-3) bp) and chromatin release. Unlike highly dispersed Alu repeat sequences, the centromeric heterochromatin was much less susceptible to endonuclease attack. In addition to chromatin release, the permeability of the sperm membrane was altered as evidenced by reduced accessibility of sperm nuclei to decondensation factors in mouse embryo extracts. Hybridization of cDNA microarrays with DNA released from spermatozoa revealed a consistent hypersensitivity of certain genes to endogenous cleavage including TP53, VHL (tumour suppressors), BRCA1 (breast cancer), NOS1 (neurotransmitter), PECAM1, FLT1 (angiogenesis) and CDKN1C (cell cycle/imprinted). N-tert-butyl hydroxylamine (NTBH), a derivative of the anti-teratogenic alpha-phenyl-N-t-butyl nitrone (PBN) and synthetic superoxide dismutase (SOD)/catalase mimetics inhibited chromatin release and sustained or dissipated relative mitochondrial membrane potential. Together, these results show a link between the hyperactivation of sperm mitochondria and chromosomal damage of specific genes in vitro, and that the potential risk of disruption of paternally contributed genes can be circumvented by antioxidants which are known to target mitochondria.
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PMID:Gene-specific chromatin damage in human spermatozoa can be blocked by antioxidants that target mitochondria. 1465 2

Epidemiological studies have indicated that ataxia-telangiectasia (AT) heterozygotes in AT families have an increased risk of cancer, particularly of breast cancer (BC). However, in BC case-control studies, no significant differences were found in the frequency of ATM mutations between patients and controls. In such studies missense mutations were found more frequently than truncating mutations, suggesting that the cancer risk depends on mutation type. To investigate this possibility, we assessed the risk of BC according to the type and position of the ATM truncating mutation in extended AT families. DNA or RNA that had been isolated from blood or buccal cells of AT children and their relatives was screened for ATM germ-line mutations using restriction endonuclease fingerprinting, the protein truncation test, fluorescence-assisted mismatch analysis, and direct sequencing. The standardized incidence ratio of cancer associated with ATM heterozygosity status and type of mutation was estimated. We tested for genotype-phenotype correlations by simulations, permuting mutations among parental branches. No significant difference was found in the relative risk of breast cancer or any other type of cancer based on mutation type. However, the occurrence of BC may be associated with truncating mutations in certain binding domains of the ATM protein (e.g., P53/BRCA1, beta-adaptin, and FAT domains; P = 0.006). In this limited sample set, the presence of missense or truncating ATM mutations was not associated with different cancer risks. The risk of BC appeared to be associated with the alteration of binding domains rather than with the length of the predicted ATM protein.
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PMID:Cancer risk according to type and location of ATM mutation in ataxia-telangiectasia families. 1539 Jan 80

As sequence analysis for BRCA1 and BRCA2 mutations is both time- and cost-intensive, current strategies often include scanning techniques to identify fragments containing genetic sequence alterations. However, a systematic assessment of the diagnostic accuracy has been lacking so far. Here, we report on a systematic review to assess the internal and external validity of current scanning techniques. Inclusion criteria were: controlled design, investigators blinded, and tests suitable as a scanning tool for the whole genes BRCA1 and BRCA2. Outcome parameters were sensitivity, specificity, and positive and negative predictive values compared to direct sequencing. Out of 3816 publications, 10 studies reporting on 12 methods met our inclusion criteria. The internal and external validity of most of these studies was limited. Sensitivities were reported to be 100% for enzymatic mutation detection (EMD), multiple-dye cleavase fragment length polymorphism (MD-CFLP), fluorescence-based conformation-sensitive gel electrophoresis (F-CSGE), RNA-based sequencing, restriction endonuclease fingerprinting-single strand conformation polymorphism (REF-SSCP), stop codon (SC) assay, and denaturing high-performance liquid chromatography (DHPLC). Sensitivity was 50-96% for SSCP, 88-91% for two-dimensional gene scanning (TDGS), 76% for conformation-sensitive gel electrophoresis (CSGE), 75% for protein truncation test (PTT), and 58% for micronucleus test (MNT). Specificities close to 100% were reported, except for MNT. PTT and SC assay are only able to detect truncating mutations. Most studies were designed to introduce new experimental approaches or modifications of established methods and require further evaluation. F-CSGE, REF-SSCP, RNA-based sequencing, EMD, and MD-CFLP will need further evaluation before their use in a routine setting can be considered. SSCP, MNT, PTT, CSGE, and TDGS cannot be recommended because of their low sensitivity. DHPLC outperforms all other methods studied. However, none of the four studies evaluating DHPLC was performed on BRCA2.
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PMID:Diagnostic accuracy of methods for the detection of BRCA1 and BRCA2 mutations: a systematic review. 1734 52

Adapter-ligation-mediated allele-specific amplification (ALM-ASA) is a potential method for multiplex SNPs typing at an ultra low cost. Here, we describe a kind of software, which designs allele-specific primers for ALM-ASA assay on multiplex SNPs. DNA sequences containing SNPs of interest are submitted into the software which contains various endonucleases for options. Based on the SNP sequence information and the selected endonucleases, the software is capable of automatically generating sets of information needed to perform genotyping experiments. Each set contains a suitable endonuclease, qualified allele-specific primers with orientations and melting temperatures, sizes of allele-specific amplicons, and gel electropherograms simulated according to the sizes of the allele-specific amplicons and the mobility of DNA fragments in 2% agarose gel. Seven SNPs in the arylamines N-acetyltransferase 2 (NAT2) gene, five SNPs in the BRCA1 gene, five SNPs in the COMT gene, six SNPs in the CYP2E1 gene, five SNPs in the MPO gene, and six SNPs in the NRG1 gene were selected for evaluating the software. Without extra optimization, seven SNPs in the NAT2 gene were successfully genotyped for genomic DNA samples from 127 individuals by using the first set of allele-specific primers yielded by the software. Although several steps are used in the ALM-ASA assay, the whole genotyping process can be completed within 3 h by optimizing each step. Profiting from the software, the ALM-ASA assay is easy-to-perform, labor-saving, and accurate.
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PMID:Improved adapter-ligation-mediated allele-specific amplification for multiplex genotyping by using software. 1831 48


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