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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two proteins, which may be involved in DNA replication, have been isolated and characterized from the eukaryote Tetrahymena thermophila. One of these proteins, DNA polymerase, has been purified to apparent homogeneity. The enzyme has a native molecular weight of approximately 90 000 in the presence of salt and aggregates to higher-molecular-weight forms in the absence of salt. Purified preparations of the enzyme yield a major subunit of Mr 45 000 when the protein is analyzed by denaturing electrophoresis. Tetrahymena DNA polymerase requires a divalent cation for catalysis and prefers gapped template-primers over denatured and native DNAs. A template-primer such as poly(dT) . oligo(A) can also be elongated by the DNA polymerase. However, the enzyme will not use poly(A) . oligo(dT) as a template-primer. Sulfhydryl-blocking reagents, such as N-ethylmaleimide, inhibit Tetrahymena DNA polymerase. The DNA polymerase lacks assayable levels of both single and double-stranded deoxyribonuclease activity. Throughout the early stages of purification the DNA polymerase chromatographs together with a protein of molecular weight 100 000. This protein, which yields a single major polypeptide of Mr 25 000 when analyzed by denaturing electrophoresis, has single-stranded-DNA-binding properties and has the ability to stimulate both the rate and extent of DNA-polymerase-catalyzed DNA synthesis in vitro. By virtue of this latter ability, the protein has been referred to as the M (for 'modifying') protein. Maximum stimulation of DNA polymerase was achieved with template-primers, which contained large stretches of single-stranded template such as poly(dA) . (dT)10 mixed in a template-to-primer ratio of one to one. Stimulation of DNA polymerase activity by M protein in vitro appears to involve formation of longer product DNA.
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PMID:Purification from Tetrahymena thermophila of DNA polymerase and a protein which modifies its activity. 746 Sep 43

Ferric nitrilotriacetate (Fe(3+)-NTA) catalyzes hydrogen peroxide-derived production of hydroxyl radicals, which are known to cause DNA damage. In the present work, Fe(3+)-NTA plus hydrogen peroxide-induced single-strand DNA breaks and repair of the DNA damage were studied in vitro by monitoring DNA damage- and DNA repair-dependent conformational changes of pUC18 plasmid DNA. Single-strand DNA breaks were induced in the pUC18 DNA by Fe(3+)-NTA plus hydrogen peroxide in a dose-dependent fashion. Induction of the DNA damage was inhibited by deferoxamine mesylate (an iron chelator) and by hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO), D-mannitol and ethanol indicating that the DNA damage was caused by hydroxyl radicals which were generated by reaction of Fe(3+)-NTA with hydrogen peroxide. The oxygen radical-induced single-strand DNA breaks were repaired partly (more than 50%) by incubating the damaged DNA at 37 degrees C for 3 h with a partially purified preparation of APEX nuclease (a multifunctional DNA repair enzyme), DNA polymerase beta, four deoxyribonucleoside triphosphates, T4 DNA ligase and ATP. Analyses of the partially purified preparation of APEX nuclease revealed that a 45-kDa protein as well as APEX nuclease in the preparation were involved in the repair of the single-strand DNA breaks. APEX nuclease was suggested to initiate the repair by removing 3' termini blocked by the nucleotide fragments and also by incising the 5' side of AP sites. The 45-kDa protein was suggested to be required for removal of the 5' tags such as 5'-terminal deoxyribose phosphate residues produced by the action of APEX nuclease on AP sites.
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PMID:Oxygen radical-induced single-strand DNA breaks and repair of the damage in a cell-free system. 756 64

Endonuclease G (Endo G) is widely distributed among animals and cleaves DNA at double-stranded (dG)n.(dC)n and at single-stranded (dC)n tracts. Endo G is synthesized as a propeptide with an amino-terminal presequence that targets the nuclease to mitochondria. Endo G can also be detected in extranucleolar chromatin. In addition to deoxyribonuclease activities, Endo G also has ribonuclease (RNase) and RNase H activities and specifically cleaves mouse mitochondrial RNA and DNA-RNA substrates containing the origin of heavy-strand DNA replication (OH). The cleavage sites match those found in vivo, indicating that Endo G is capable of generating the RNA primers required by DNA polymerase gamma to initiate replication of mitochondrial DNA.
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PMID:Primers for mitochondrial DNA replication generated by endonuclease G. 768 44

Drosophila Rrp1 (Recombination repair protein 1) belongs to a family of DNA repair nucleases that includes Escherichia coli exonuclease III, Streptococcus pneumoniae exonuclease A, bovine BAP, mouse APEX endonuclease, and human APE. Within a 252 amino acid region, colinear homology is shared between all members. Rrp1 is unique in that it includes a 427 amino acid N-terminal region not related to any known sequence. The protein copurifies with an apurinic endonuclease and a double-stranded DNA 3'-exonuclease. In this study, a 5'-end-labeled 37 base pair oligonucleotide substrate containing a single apurinic site was used to characterize the endonuclease activity of Rrp1. This substrate is utilized efficiently by Rrp1: the specific activity observed is 1 x 10(5) units/mg. The abasic double-stranded DNA oligonucleotide is cleaved only at the abasic site to create a single-strand break. Strand breaks are not detected in the complementary strand, in the single-stranded DNA oligonucleotide, or in the base-paired control substrate. After endonucleolytic cleavage at the abasic site, exonucleolytic processing at the nick is slow and requires a molar excess of Rrp1, while exonuclease III degrades the nicked substrate more efficiently. The Rrp1 cleavage product comigrates with a DNaseI cleavage product, and the newly formed terminus supports DNA synthesis by DNA polymerase. Therefore, Rrp1 cleaves the phosphodiester backbone at one position 5' to the apurinic site and leaves a 3'-hydroxyl terminus. Rrp1 is a class II apurinic endonuclease and is likely to be important in DNA repair in Drosophila.
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PMID:Characterization of the apurinic endonuclease activity of Drosophila Rrp1. 769 63

The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.
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PMID:DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract. 782 35

A comparison was carried out for the Km and maximal rates of conversion on (Vmax) with primers containing noncomplementary bases, as well as primers without several bases, and fully complementary primers of the same length. The number of complementary bases from the 3' end to the noncomplementary nucleotide was shown to determine the efficiency of interaction (and conversion) of the primers containing noncomplementary bases with enzyme. The DNA polymerase practically does not discriminate between the primers without one or two bases and the fully complementary primers. Elimination of one base in any position from the 3' end of the primer is equivalent to shortening of the primer by one nucleotide unit, and leads to a decrease in the affinity by a factor of 1.8. We suppose that DNA polymerase does not participate in primer mistake correction during the repair process if the DNA contains apurinic or apyrimidinic nucleotide units.
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PMID:[Recognition of primers, containing noncomplementary nucleotides and units, lacking bases, by the Klenow fragment of Escherichia coli DNA polymerase I]. 828 84

The replication of Epstein-Barr virus (EBV) and the expression of EBV early proteins were studied in the Burkitt's lymphoma cell line Akata stimulated with anti-human immunoglobulin G antibody (anti-IgG). Akata cells contained approximately 20 copies of EBV genome per cell as covalently closed, circular DNA. EBV DNA replication was observed at 6 hr and reached a maximal level at 24 hr after treatment with anti-IgG. Virion DNA was found in the culture medium at 12 hr. The kinetics of expression of BMRF1 gene product (early antigen diffuse component; EA-D) paralleled that of EBV deoxyribonuclease (DNase) and of DNA polymerase. Immunoblotting analysis showed that three polypeptides with molecular masses of 54, 52, and 49 kilodaltons (kDa) were recognized as EA-D components. The EBV DNase polypeptide was detected by immunoblotting at 53 kDa. The anti-EBV DNA polymerase antibody recognized 120- and 54-kDa polypeptides in the Akata cells. Immunoprecipitation followed by immunoblotting showed that EA-D and EBV DNase polypeptides were coimmunoprecipitated with anti-EBV DNase antibody and with anti-EA-D monoclonal antibody. These findings indicate that EA-D forms a complex with EBV DNase polypeptide. The molecules of EA-D, EBV DNase, and DNA polymerase appear to be closely associated together on the EBV replication.
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PMID:Epstein-Barr virus (EBV) replication and expressions of EA-D (BMRF1 gene product), virus-specific deoxyribonuclease, and DNA polymerase in EBV-activated Akata cells. 839 19

Exposure to exogenous alkylating agents, particularly N-nitroso compounds, has been associated with increased incidence of primary human brain tumors, while intrinsic risk factors are currently unknown. The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a major defense against the carcinogenicity of N-nitroso compounds and other alkylators. We report here that in 55% (64/117) of cases, histologically normal brain tissue adjacent to primary human brain tumors lacked detectable MGMT activity [methyl excision repair-defective (Mer-) status]. The incidence of Mer- status in normal brain tissue from brain tumor patients was age-dependent, increasing from 21% in children 0.25-19 years of age to 75% in adults over 50. In contrast, Mer- status was found in 12% (5/43) of normal brain specimens from patients operated for conditions other than primary brain tumors and was not age-dependent. The 4.6-fold elevation in incidence of Mer- status in brain tumor patients is highly significant (chi2 = 24; p < or = 0.001). MGMT activity was independent of age in the lymphocytes of brain tumor patients and was present in lymphocytes from six of nine tumor patients whose normal brain specimen was Mer-. DNA polymerase beta, apurinic/apyrimidinic endonuclease, and lactate dehydrogenase activities were present in all specimens tested, including Mer- specimens from brain tumor patients. Our data are consistent with a model of carcinogenesis in human brain in which epigenetically regulated lack of MGMT is a predisposing factor and alkylation-related mutagenesis is a driving force.
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PMID:Lack of the DNA repair protein O6-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary human brain tumors. 869 23

We purified an apurinic/apyrimidinic (AP) endonuclease from mouse ascites sarcoma (SR-C3H/He) cells. The enzyme showed nicking activity on acid-depurinated DNA but not on untreated, intact DNA. It also showed priming activity for DNA polymerase on both acid-depurinated and bleomycin-damaged DNA. The priming activity on bleomycin-damaged DNA was two times higher than that on an acid-depurinated DNA. The enzymatic properties indicate that the enzyme is a class II AP endonuclease having DNA 3' repair diesterase activity. The purified enzyme has a molecular weight of 39,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal pH for AP endonuclease activity was 8.0 in 50 mM Tris-HCl buffer. The AP endonuclease activity depended on divalent cation such as Mg2+ and Co2+ ions, and was inhibited by 2 mM EDTA with no addition of the divalent cation. An appropriate concentration of sodium or potassium salt stimulated the activity. Partial digestion of the AP endonuclease with Staphylococcus aureus V8 protease produced 4 major peptide fragments which may be used for protein sequencing.
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PMID:Purification and characterization of a 39kDa apurinic/apyrimidinic endonuclease from mouse ascites sarcoma cells. 880 52

We have established four cell lines, UW228-1, UW228-2, UW228-3 and UW443, from two posterior fossa medulloblastomas. The three UW228 sublines originated from a tumor with a diploid DNA content, while the tumor of origin of UW443 was predominantly tetraploid. Both tumors displayed areas of immunopositivity for synaptophysin and glial fibrillary acidic protein. All four cell lines have been grown as monolayers in continuous culture for 50 to 200 passages, are not contact inhibited at high density, and form colonies in soft agar. The UW228 sublines are aneuploid, have similar modal chromosome numbers, similar chromosomal duplications and identical marker chromosomes, and display loss of heterozygosity for identical sequences at the distal end of chromosome 17p. UW443 is diploid and also shows loss of heterozygosity for a distal sequence on chromosome 17p. All lines are immunopositive for two or more neurofilament proteins, three lines (UW228-1, UW228-2 and UW443) are immunopositive for synaptophysin, and none are immunopositive for glial fibrillary acidic protein. The lines differ in sensitivity to the alkylating agents 1,3-bis(2-chloroethyl)-1-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. They also differ in dependence on the DNA repair protein O6-methylguanine-DNA methyltransferase for alkylating agent resistance and in levels of the DNA repair activities apurinic/apyrimidinic endonuclease and DNA polymerase beta. These properties establish UW228-1, UW228-2, UW228-3 and UW443 as four new, phenotypically distinct medulloblastoma-derived cell lines.
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PMID:Establishment and characterization of four human medulloblastoma-derived cell lines. 886 61


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