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)

Xenopus laevis DNA polymerase gamma co-purifies with a tightly associated 3'----5' exonuclease. The purified enzyme lacks 5'----3' exonuclease and endonuclease activity. The ratio of the 3'----5' exonuclease activity to DNA polymerase gamma activity remains constant over the final three chromatographic procedures. In addition, these activities co-sediment under partially denaturing conditions in the presence of ethylene glycol. The associated 3'----5' exonuclease activity removes a terminally mismatched nucleotide more rapidly than a correctly base-paired 3'-terminal residue, as expected if this exonuclease has a proofreading function. The 3'----5' exonuclease has the ability to release a terminal phosphorothioated nucleotide, a property shared with T4 DNA polymerase, but not with Escherichia coli DNA polymerase I.
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PMID:DNA polymerase gamma from Xenopus laevis. II. A 3'----5' exonuclease is tightly associated with the DNA polymerase activity. 260 78

Mutagenesis, clastogenesis, and carcinogenesis, may all be S-phase dependent processes within carcinogen-damaged human cells. Carcinogens have been shown to inhibit replicative DNA synthesis in S phase cells and the mechanisms of inhibition have been identified. It is proposed that the sequelae of carcinogen action (mutations, sister-chromatid exchanges, chromosome aberrations) are the consequence of the production of lesions in the DNA template which interfere with the ability of DNA polymerase to synthesize a complementary strand without error. Mis-instructive lesions in the template give rise to base-substitution mutations in nascent strands as DNA polymerase inserts an incorrect but complementary base. Non-instructive base lesions and sterically interfering bulky adducts in the template inhibit DNA polymerase and cause the growing points of nascent DNA strands to be blocked. This blockage perpetuates discontinuities in daughter strands. These discontinuities are eliminated by a process known as post-replication repair. Blocked growing points may be relieved by un-directed insertion of DNA precursors to span the non-instructive lesions. Transient dislocation of the primer terminus from the damaged template may occur at palindromic or repetitive sequences. Reannealing of the primer terminus beyond the site of damage may allow bypass of blocking lesions with a consequence of deletion or insertion of genetic information. DNA at the site of blocked growing points may be a substrate for other enzymes involved in DNA metabolism. Single-strand gaps in daughter strands may be recognized by Rec A-like proteins which catalyze paranemic invasion of sister duplex strands. Recombination intermediates generated at sites of blocked growing points may be resolved by a pathway that produces either sister-chromatid exchanges or the insertion of a patch of parental template DNA within the daughter strand. Single-strand-specific endonuclease may attack regions of denatured DNA at blocked growing points producing double-strand breaks which appear to be intermediates in the formation of chromatid aberrations. The utilization of each of these pathways of post-replication repair will depend upon the precise structure of the template lesion, the sequence context in which the lesion is embedded in the template strand, and stochastic processes.
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PMID:Pathways of human cell post-replication repair. 264 48

Circular plasmid DNA is known to replicate when injected into unfertilized eggs of Xenopus laevis. We characterized early DNA synthesis which precedes the replication. Incorporation of a radioactive precursor into covalent closed circular DNA becomes detectable as early as 2 minutes after injection. Judging from the sensitivity to aphidicolin, replicative DNA polymerase alpha or delta is involved in this reaction. However, analysis of density-labeled product as well as detection of newly synthesized, unmethylated strands by restriction endonuclease DpnI digestion, both indicated that most if not all, of the synthesized DNA is present as short repair-like patches in the injected DNA molecules. They are present randomly in all the HaeIII fragments of injected DNA, and transcriptional activation of the 5S RNA gene inserted in the plasmid does not affect the distribution. Only a minor fraction of injected DNA is utilized for this reaction, and 12% of such DNA molecules are further chased into replicated progeny DNA. This efficiency of replication is nearly the same as that of bulk injected DNA which has not been subjected to the early DNA synthesis. We concluded that, despite the common use of replicative DNA polymerase, the early DNA synthesis and the subsequent DNA replication are mutually independent processes.
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PMID:Characterization of early DNA synthesis in Xenopus eggs after injection of circular plasmid DNA. 273 1

In the Mediterranean area, 50% of the beta thalassaemia mutations abolish or create a restriction endonuclease site in the beta globin gene. This study describes a new procedure for prenatal detection of these beta thalassaemia defects based on the direct visualisation, on an ethidium bromide stained polyacrylamide gel, of the discrete DNA fragments produced by restriction endonuclease digestion of fetal DNA, enzymatically amplified using the DNA polymerase from the thermophilus bacterium Thermus aquaticus. We applied this procedure to the Sardinian population to detect the nonsense mutation at codon 39 and the frameshift at codon 6 of the beta globin gene; these are the most frequent beta thalassaemia mutations in this population, accounting for 95% and 2.2% of the beta thalassaemia chromosomes. The main advantages of this procedure are simplicity (no radioactivity), sensitivity (0.2 microgram of DNA), and rapidity (12 hours). The very small amount of fetal material required makes amniotic fluid cell culture unnecessary and may decrease the fetal loss rate associated with trophoblast sampling. By circumventing the use of radioactive and non-radioactive probes, the spread of this technology to the high risk areas will be facilitated.
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PMID:Prenatal diagnosis of beta thalassaemia based on restriction endonuclease analysis of amplified fetal DNA. 273 98

The value of the unscheduled DNA synthesis after the combined effect of UV radiation and methyl methanesulfonate (MMS) was considerably lower than that upon exposure to UV radiation alone and after two-hour incubation of the culture. These differences were insignificant after 26 h incubation. The result can be attributed to the alkylating effect of MMS on the repair DNA polymerase. With MMS delivered prior to UV irradiation there was an even larger decrease in the unscheduled DNA synthesis with both 2- and 26-hour incubation. The data obtained can be explained by the fact that MMS inhibits an excision endonuclease.
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PMID:[The modifying action of methylmethane sulfonate on unscheduled DNA synthesis in the UV irradiation of human peripheral blood lymphocytes]. 278 Sep 92

Three major polypeptides are found in purified DNA polymerase alpha from rat liver: 160, 77 and 58 kDa. The electrophoretic analysis has identified polypeptide 160 kDa as the catalytically active subunit of DNA polymerase alpha. The other two polypeptides showed no DNA polymerase activity. Individual polypeptide p77 kDa purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to produce antibodies in rabbits. Immunoblot analysis indicated that the complex DNA polymerase alpha-3'-5'-exonuclease contained polypeptide p77 kDa. To elucidate the function of the p77 kDa protein we have prepared an immunoabsorbent column with antibodies against the p77 kDa polypeptide. The antibody column purified p77 kDa protein was homogeneous according to sodium dodecyl sulfate gel electrophoresis. The activity of alpha-polymerase was increased approximately 10-fold as a result of purification of DNA polymerase alpha from the p77 kDa protein. The in vitro experiments showed the identity of the p77 kDa polypeptide to endonuclease. It cleaved both single-stranded and double-stranded DNA. The function of endonuclease p77 kDA in complex with DNA polymerase alpha remains obscure.
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PMID:[Isolation and characteristics of endonuclease tightly bound to alpha-polymerase from the rat liver]. 284 24

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.
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PMID:Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma. 296 5

Ultraviolet light irradiation of DNA results in the formation of two major types of photoproducts, cyclobutane dimers and 6-4' [pyrimidin-2'-one] -pyrimidine photoproducts. The enzyme T4 DNA polymerase possesses a 3' to 5' exonuclease activity and hydrolyzes both single and double stranded DNA in the absence of deoxynucleotide triphosphate substrates. Here we describe the use of T4 DNA polymerase associated exonuclease for the detection and quantitation of UV light-induced damage on both single and double stranded DNA. Hydrolysis of UV-irradiated single or double stranded DNA by the DNA polymerase associated exonuclease is quantitatively blocked by both cyclobutane dimers and (6-4) photoproducts. The enzyme terminates digestion of UV-irradiated DNA at the 3' pyrimidine of both cyclobutane dimers and (6-4) photoproducts. For a given photoproduct site, the induction of cyclobutane dimers was the same for both single and double stranded DNA. A similar relationship was also found for the induction of (6-4) photoproducts. These results suggest that the T4 DNA polymerase proofreading activity alone cannot remove these UV photoproducts present on DNA templates, but instead must function together with enzymes such as the T4 pyrimidine dimer-specific endonuclease in the repair of DNA photoproducts. The T4 DNA polymerase associated exonuclease should be useful for the analysis of a wide variety of bulky, stable DNA adducts.
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PMID:T4 DNA polymerase (3'-5') exonuclease, an enzyme for the detection and quantitation of stable DNA lesions: the ultraviolet light example. 298 81

We determined the complete nucleotide sequence of the intracisternal A-particle gene, IAP-H18, cloned from the normal Syrian hamster liver DNA. IAP-H18 was 7,951 base pairs in length with two identical long terminal repeats of 376 base pairs at both ends. On the coding strand, imperfect open reading frames corresponding to gag and pol of the retrovirus genome were observed, whereas many stop codons were present in the region corresponding to env. The putative H18 gag gene (809 amino acids) had a sequence homologous to the N-terminal half of the mouse mammary tumor virus gag gene and locally to the Rous sarcoma virus gag gene. The putative H18 pol gene (900 residues) was homologous to the Rous sarcoma virus pol gene almost throughout the entire region. Two conserved regions among the retrovirus pol genes have been reported. One presumably corresponds to the DNA polymerase and the RNase H domain, and the other corresponds to the DNA endonuclease domain of the multifunctional protein pol. By the comparison of the deduced amino acid sequences of the putative endonuclease domain of six representative oncovirus genomes, a phylogenetic tree of the oncovirus genomes was constructed, and the intracisternal A-particle (type A) genome was found to be more closely related to the mouse mammary tumor virus (type B) and squirrel monkey retrovirus (type D) genomes.
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PMID:Nucleotide sequence of the Syrian hamster intracisternal A-particle gene: close evolutionary relationship of type A particle gene to types B and D oncovirus genes. 299 63

A procedure has been designed for changing specific nucleotides in a DNA sequence with efficiency. The method involves the use of the specially constructed cloning vectors pBRS1, pHS1, and pHS2. These plasmids are derivatives of pBR322 in which the EcoRI-HindIII region has been replaced by synthetic duplexes carrying SmaI, HpaI and XhoI sites, in addition to EcoRI and HindIII sites. The DNA fragment to be mutagenized is cloned in pHS1 (or pBRS1, or pHS2) using restriction sites close to the SmaI and HpaI sites. The recombinant plasmid obtained is digested with one of these enzymes to produce double-stranded DNA with blunt ends. This linear DNA is a substrate for exonuclease III (or T4 DNA polymerase). The digestion under controlled conditions produces duplex with protruding single-stranded 5'-regions which include the site of the desired mutation. The synthesis of DNA by DNA-polymerase I (Klenow's fragment), primed in part by the synthetic oligonucleotide containing the desired mutation, leads to the linear heteroduplex. The closed circular heteroduplex is formed by ligation. After transformation into E. coli, DNA replication generates homoduplexes, some of which contain the mutation. Colony hybridization with the same 32P-labeled oligonucleotide is used to select mutants. The yield of the mutants is 10-20%. This technique can be extended to replicative form of M13 vectors. It can be also applied to any DNA sequence which has a unique site of restriction endonuclease generating blunt ends.
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PMID:[Effective method of oligonucleotide-controlled mutagenesis of DNA fragments]. 299 83


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