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 second enzyme in the DNA base excision repair (BER) pathway, apurinic/apyrimidinic (AP) endonuclease or Ape1, hydrolyzes the phosphodiester backbone immediately 5' to an AP site generating a normal 3'-hydroxyl group and an abasic deoxyribose-5-phosphate, which is processed by subsequent enzymes of the BER pathway. AP sites are the most common form of DNA damage, and the persistence of AP sites in DNA results in a block to DNA replication, cytotoxic mutations, and genetic instability. Interestingly, Ape1/ref-1 is a multifunctional protein that not only is a DNA repair enzyme, but also functions as a redox factor maintaining transcription factors, such as Fos, Jun, nuclear factor-kappaB, PAX (paired box-containing family of genes), hypoxia inducible factor-lalpha (HIF-1alpha), HIF-1-like factor, and p53, in an active reduced state. Apel/ref-1 has also been implicated in a number of other activities, one of which is the activation of bioreductive drugs requiring reduction for activity. In this report, we present data supporting our findings that another level of posttranslational modification of Apel/ref-1 that clearly affects the AP endonuclease activity is the reduction or oxidation of this protein. Furthermore, we show data demonstrating that at least one of the sites involved in this redox regulation is the cysteine amino acid found at position 310, immediately adjacent to the crucial histidine residue at position 309 in the DNA repair active site. These findings suggest that the Apel/ref-1 protein may be much more intimately regulated at the posttranslational level than initially imagined.
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PMID:Redox regulation of the DNA repair function of the human AP endonuclease Ape1/ref-1. 1155 53

Knowledge of inherited and sporadic mutations in known and candidate cancer genes may influence clinical decisions. We have developed a mutation scanning method that combines thermostable EndonucleaseV (Endo V) and DNA ligase. Variant and wild-type PCR amplicons are generated using fluorescently labeled primers, and heteroduplexed. Thermotoga maritima (Tma) EndoV recognizes and primarily cleaves heteroduplex DNA one base 3' to the mismatch, as well as nicking matched DNA at low levels. Thermus species (Tsp.) AK16D DNA ligase reseals the background nicks to create a highly sensitive and specific assay. The fragment mobility on a DNA sequencing gel reveals the approximate position of the mutation. This method identified 31/35 and 8/8 unique point mutations and insertions/deletions, respectively, in the p53, VHL, K-ras, APC, BRCA1, and BRCA2 genes. The method has the sensitivity to detect K-ras mutations diluted 1 : 20 with wild-type DNA, a p53 mutation in a 1.7 kb amplicon, and unknown p53 mutations in pooled DNA samples. EndoV/Ligase mutation scanning combined with PCR/LDR/Universal array proved superior to automated DNA sequencing for detecting p53 mutations in colon tumors. This technique is well suited for scanning low-frequency mutations in pooled samples and for analysing tumor DNA containing a minority of the unknown mutation.
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PMID:An endonuclease/ligase based mutation scanning method especially suited for analysis of neoplastic tissue. 1189 24

Mutations in the mismatch DNA repair gene human MutS homologen 2 (hMSH2) are causative for microsatellite instability and carcinogenesis in various human tumours, including hereditary nonpolyposis colorectal cancer. Because microsatellite instability has been detected in malignant melanoma, we have investigated hMSH2 in melanocytic tumours. We found strong nuclear immunoreactivity for hMSH2 that was elevated in malignant melanoma and melanoma metastases as compared to acquired nevi. These findings suggest that increased genomic instability in malignant melanoma is associated with elevated protein levels of this DNA repair enzyme. hMSH2 is not exclusively regulated by proliferative activity in melanocytes, because there was no correlation between staining patterns of hMSH2 and the proliferation marker Ki-67. In contrast, immunoreactivity scores for hMSH2 and p53 were both upregulated in malignant melanocytic tumours. These findings support the concept that hMSH2 gene expression may be regulated in melanocytes by the p53 protein, as has been reported previously in other tissues. Using the reverse transcription-polymerase chain reaction, we detected strong hMSH2 mRNA expression in each of 8 melanoma cell lines analysed (highest amounts in SK-MEL-25 cells, lowest amounts in MML-I cells). In conclusion, our findings indicate that hMSH-2 may be of importance for genetic stability, tumorigenesis and progression of malignant melanoma.
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PMID:DNA mismatch repair enzyme hMSH2 in malignant melanoma: increased immunoreactivity as compared to acquired melanocytic nevi and strong mRNA expression in melanoma cell lines. 1193 86

Balanced regulation of DNA double-strand break (DSB) repair is crucial for genetic integrity and cell survival. Cells perform DSB repair either by homologous recombination (HR) or by non-homologous end joining (NHEJ). Either option carries risk for DNA instability. The presence in the cell of the tumour suppressor p53 has been shown to suppress the levels of HR; however, the effect of p53 on DNA EJ is less well understood. Here we demonstrate dramatically increased DNA EJ activity in cell-free extracts from p53(-/-) mouse embryo fibroblasts (MEFs) compared with p53(+/+) MEFs. The addition of wild-type (wt) p53 to p53(-/-) MEFs extracts inhibited DNA EJ in a dose-dependent manner. Binding of wt p53 to DNA ends in vitro protected them from exonuclease attack and inhibited T4 DNA ligase-dependent EJ. This inhibitory effect was markedly enhanced for p53 R175H, a cancer-derived mutant of p53. In contrast, inhibition was negated in the presence of p53 S15D, a phosphorylation-mimicking mutant protein. Interestingly, p53 S15D stimulated in vitro DNA EJ of the blunt-ended DNA by T4 DNA ligase. Here we discuss the possibility that, in conjunction with its ability to control levels of HR, p53 may also serve to suppress DNA EJ in cells under normal conditions. This suppression may be associated with DNA-dependent protein kinases or ATM kinases, providing potential crosstalk between major cellular pathways of DNA repair and cell-cycle checkpoint mechanisms.
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PMID:Effect of wild-type, S15D and R175H p53 proteins on DNA end joining in vitro: potential mechanism of DNA double-strand break repair modulation. 1196 Sep 5

The mutagenicity of a prominent tobacco carcinogen, benzo[a]pyrene (B[a]P), is believed to result from chemical reactions between its diol epoxide metabolite, (+)-anti-7r,8t-dihydroxy-c9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and DNA, producing promutagenic lesions, e.g., (+)-trans-anti-7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (N(2)-BPDE-dG). Previous studies used the DNA repair enzyme UvrABC endonuclease in combination with ligation-mediated PCR (LMPCR) to demonstrate an increased reactivity of BPDE toward guanine nucleobases within codons 157, 248, and 273 of the p53 tumor suppressor gene (Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. Science 274, 430-432). These sites are also "hot spots" for mutations observed in lung tumors of smokers, suggesting an involvement of B[a]P in the initiation of lung cancer. However, the LMPCR approach relies on the ability of the repair enzyme to excise BPDE-induced lesions, and thus the slowly repaired lesions may escape detection. Furthermore, BPDE-DNA adduct structure and stereochemistry cannot be determined. In the present work, we performed a direct quantitative analysis of N(2)-BPDE-dG originating from specific guanine nucleobases within p53- and K-ras-derived DNA sequences by using a stable isotope labeling-mass spectrometry approach recently developed in our laboratory. (15)N-labeled dG was placed at defined positions within DNA sequences derived from the K-ras proto-oncogene and p53 tumor suppressor gene, the two genes most frequently mutated in smoking-induced lung cancer. (15)N-labeled DNA was annealed to the complementary strands, followed by BPDE treatment and liquid chromatography-electrospray ionization tandem mass spectrometry analysis (HPLC-ESI-MS/MS) of N(2)-BPDE-dG lesions. The extent of adduct formation at (15)N-labeled guanine was determined directly from the HPLC-ESI-MS/MS peak area ratios of (15)N-N(2)-BPDE-dG and N(2)-BPDE-dG. BPDE-induced guanine adducts were produced nonrandomly along K-ras and p53 gene-derived DNA sequences, with over 5-fold differences in adduct formation depending on sequence context. N(2)-BPDE-dG yield was enhanced by the presence of 5-Me substituent at the cytosine base-paired with the target guanine nucleobase, an endogenous DNA modification characteristic for CpG dinucleotides within the p53 gene. In the K-ras-derived DNA sequence, the majority of N(2)-BPDE-dG adducts originated from the first position of the codon 12 (GGT), consistent with the large number of G --> T transversions observed at this nucleotide in smoking-induced lung cancer. On the contrary, the pattern of N(2)-BPDE-dG formation within the p53 exon 5 sequences did not correlate with the mutational spectrum in lung cancer, suggesting that factors other than N(2)-BPDE-dG formation are responsible for these mutations. The stable isotope labeling HPLC-ESI-MS/MS approach described in this work is universally applicable to studies of modifications to isolated DNA by other carcinogens and alkylating drugs.
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PMID:Formation of benzo[a]pyrene diol epoxide-DNA adducts at specific guanines within K-ras and p53 gene sequences: stable isotope-labeling mass spectrometry approach. 1213 76

The DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) removes alkylating adducts from the O(6) position of guanine and protects cells from cytotoxic and mutagenic effects. Expression of MGMT is decreased in some cancers, which may be the result of methylation of CpG islands of both the promoter and coding regions of the gene. We studied the methylation status of the MGMT promoter in a very large collection of brain tumors (85) using methylation-specific polymerase chain reaction (PCR). Aberrant methylation occurred in 48% of 85 human brain tumor samples. Quantitative real-time PCR showed that expression of MGMT mRNA levels was significantly decreased (P < 0.001) in those brain tumors that had methylation of the promoter region of their MGMT gene. MGMT can prevent G to A mutations by removing alkyl groups from the O(6) position of guanine. We found a significantly increased frequency of G:C to A:T mutations of the p53 gene in brain tumors having a methylated MGMT promoter compared with those having an unmethylated MGMT promoter (P < 0.05), and all the non-CpG dinucleotide G:C to A:T mutations of p53 were in samples with a methylated MGMT promoter.
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PMID:DNA repair gene O6-methylguanine-DNA methyltransferase: promoter hypermethylation associated with decreased expression and G:C to A:T mutations of p53 in brain tumors. 1250 76

Anaesthetics have gained a lot of attention for their potential mutagenic/carcinogenic effects. In the present study we have investigated the genotoxicity of the inhalation anaesthetic sevoflurane on DNA of lymphocytes isolated from 20 patients undergoing orthopaedic surgery. The genotoxicity of the anaesthetic was studied by assaying DNA damage, apoptosis, DNA repair enzyme activity and GSH content in peripheral lymphocytes before, 15 min after anaesthesia and 24 h after surgery. Lymphocytes isolated 15 min after anaesthesia showed an increase in oxidized purine and pyrimidine bases without DNA strand break formation. DNA strand breaks occurred on the first post-operative day, associated with an enhancement of DNA repair activity and a decrease in GSH. Formation of strand breaks could be the consequence of DNA repair activity. In fact, at 24 h after surgery most of the oxidized DNA bases were repaired. When DNA damage was not repaired, activation of the cell cycle checkpoint protein p53 could lead to apoptosis. An altered redox status may contribute to lymphocytopenia due to an apoptotic event as a consequence of surgical trauma. The presence of apoptotic cells at 1 day after surgery could support the hypothesis that highly damaged peripheral lymphocytes are committed to undergo programmed cell death if the damage is not repaired. In conclusion, the actual risk from anaesthesia is presumably extremely small. However, these findings contribute to our understanding of the regulation of DNA damage/repair and cell death.
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PMID:Lymphocyte DNA damage precedes DNA repair or cell death after orthopaedic surgery under general anaesthesia. 1296 Apr 10

Although clustered DNA damages are induced in cells by ionizing radiation and can be induced artifactually during DNA isolation, it was not known if they are formed in unirradiated cells by normal oxidative metabolism. Using high-sensitivity methods of quantitative gel electrophoresis, electronic imaging, and number average length analysis, we found that two radiosensitive human cell lines (TK6 and WI-L2-NS) accumulated Fpg-oxidized purine clusters and Nth-oxidized pyrimidine clusters but not Nfo-abasic clusters. However, four repair-proficient human lines (MOLT 4, HL-60, WTK1, and 28SC) did not contain significant levels (<5/Gbp) of any cluster type. Cluster levels were independent of p53 status. Measurement of glycosylase levels in 28SC, TK6, and WI-L2-NS cells suggested that depressed hOGG1 and hNth activities in TK6 and WI-L2-NS could be related to oxybase cluster accumulation. Thus, individuals with DNA repair enzyme deficiencies could accumulate potentially cytotoxic and mutagenic clustered DNA damages. The absence of Nfo-detected endogenous clusters in any cells examined suggests that abasic clusters could be a signature of cellular ionizing radiation exposure.
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PMID:Are endogenous clustered DNA damages induced in human cells? 1525 20

The ability to associate mutations in cancer genes with the disease and its subtypes is critical for understanding oncogenesis and identifying biomarkers for clinical diagnosis. A two-step mutation scanning method that sequentially used endonuclease V (EndoV) to nick at mismatches and DNA ligase to reseal incorrectly or nonspecifically nicked sites was previously developed in our laboratory. Herein we report an optimized single-step assay that enables ligase to proofread EndoV cleavage in real-time under a compromise between buffer conditions. Real-time proofreading results in a dramatic reduction of background cleavage. A universal PCR strategy that employs both unlabeled gene-specific primers and labeled universal primers, allows for multiplexed gene amplification and precludes amplification of primer dimers. Internally labeled PCR primers eliminate EndoV cleavage at the 5' terminus, enabling high-throughput capillary electrophoresis readout. Furthermore, signal intensity is increased and artifacts are reduced by generating heteroduplexes containing only one of the two possible mismatches (e.g. either A/C or G/T). The single-step assay improves sensitivity to 1:50 and 1:100 (mutant:wild type) for unknown mutations in the p53 and K-ras genes, respectively, opening prospects as an early detection tool.
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PMID:High sensitivity EndoV mutation scanning through real-time ligase proofreading. 1551 9

Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first-line treatment of glioblastoma and which also has significant activity in recurrent disease. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O6-alkylating agent-based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL-X(L). We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clonogenic survival after TMZ exposure are highly correlated (p < 0.0001, r2 = 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post-meiotic segregation increased 2. The MGMT inhibitor O6-benzylguanine sensitizes MGMT-positive glioma cells to TMZ whereas MGMT gene transfer into MGMT-negative cells confers protection. The antiapoptotic BCL-X(L) protein attenuates TMZ cytotoxicity in MGMT-negative LNT-229 but not in MGMT-positive LN-18 cells. Neither ionizing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild-type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild-type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ.
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PMID:O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells. 1640 12


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