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Query: UNIPROT:P06889 (Mol)
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The reactivity of endonuclease VII (gp49 of phage T4) with DNA-loops of eight, four, or one nucleotide, or any of 12 possible base mismatches was tested in vitro. Endonuclease VII introduces double-strand breaks by nick and counter-nick within six nucleotides 3' from the mispairings. High relative cleavage efficiencies at mismatches in heteroduplexes correlate with their decreased thermal stability and vice versa. A delay between nick and counter-nick was sufficient to allow T4 DNA-polymerase and T4 DNA-ligase to correct a C/C-mismatch in vitro, thereby saving the DNA from double-strand breakage. Very short repair tracks of three to four nucleotides mapped between the mismatch and one of the formerly induced nicks, which were subsequently sealed by DNA ligase.
J Mol Biol 1993 Apr 05
PMID:Endonuclease VII of phage T4 triggers mismatch correction in vitro. 847 39

The Schizosaccharomyces pombe rad1+ gene is involved in the G2 DNA damage cell-cycle checkpoint and in coupling mitosis to completed DNA replication. It is also required for viability when the cdc17 (DNA ligase) or wee1 proteins are inactivated. We have introduced mutations into the coding regions of rad1+ by site-directed mutagenesis. The effects of these mutations on the DNA damage and DNA replication checkpoints have been analyzed, as well as their associated phenotypes in a cdc17-K42 or a wee1-50 background. For all alleles, the resistance to radiation or hydroxyurea correlates well with the degree of functioning of checkpoint pathways activated by these treatments. One mutation, rad1-S3, completely abolishes the DNA replication checkpoint while partially retaining the DNA damage checkpoint. As single mutants, the rad1-S1, rad1-S2, rad1-S5, and rad1-S6 alleles have a wild-type phenotype with respect to radiation sensitivity and checkpoint functions; however, like the rad1 null allele, the rad1-S1 and rad1-S2 alleles exhibit synthetic lethality at the restrictive temperature with the cdc17-K42 or the wee1-50 mutation. The rad1-S5 and rad1-S6 alleles allow growth at higher temperatures in a cdc17-K42 or wee1-50 background than does wild-type rad1+, and thus behave like "superalleles." In most cases both chromosomal and multi-copy episomal mutant alleles have been investigated, and the agreement between these two states is very good. We provide evidence that the functions of rad1 can be dissociated into three groups by specific mutations. Models for the action of these rad1 alleles are discussed. In addition, a putative negative regulatory domain of rad1 is identified.
Mol Biol Cell 1995 Dec
PMID:Separation of phenotypes in mutant alleles of the Schizosaccharomyces pombe cell-cycle checkpoint gene rad1+. 859 Aug 6

The mutations spectra of cis-syn, trans-syn-I, (6-4), and Dewar pyrimidone photoproducts of the TT site of AATTAA and TATTAT in the (-) strand of a heteroduplex M13 vector were obtained in an excision and photoreversal repair deficient Escherichia coli host under SOS conditions. Oligonucleotides containing site-specific photoproducts were annealed to a complementary uracil-containing (+) strand that contained one or more unique pairs of nucleotide mismatches and used to prime (-) strand synthesis with a DNA polymerase and dNTPs. Following DNA synthesis, the reaction mixtures were incubated with T4 DNA ligase and ATP and then used to transfect SOS-induced competent CSRO6F' cells (uvrA6 and phr-1). The transfectants were plated, gridded, and probed by oligonucleotides specific for progeny of the (-) and (+) strands. Individual progeny of the photoproduct-containing (-) strands were plaque purified and sequenced by the dideoxy method. The cis-syn and trans-syn-I dimers were found not to be very mutagenic (<9%), the Dewar product more so (<33%), and the (6-4) product the most mutagenic (<73%). The mutation spectra were similar to those previously reported for the same photoproducts of the TT site of AGTTGG in the (+) strand of an M13 vector [Lawrence, C. W., et al. (1990) Mol. Gen Genet. 222, 166-168; LeClerc, J. E., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9685-9689] except that -1 deletion mutations were not observed for the trans-syn-I photoproducts, and a lower frequency of 3'-T-->C mutations was observed for the (6-4) photoproduct. Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side of the photoproduct. Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side was obtained from transfection experiments in which a second double mismatch was introduced opposite or flanking the photoproduct. Analysis of the minor tandem mutations induced by the (6-4) and Dewar products suggests that the SOS polymerase complex is able to elongate what amounts to double mismatches opposite these photoproducts and is consistent with the action of a highly processive polymerase that lacks proofreading ability.
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PMID:Mutation spectra of M13 vectors containing site-specific Cis-Syn, Trans-Syn-I, (6-4), and Dewar pyrimidone photoproducts of thymidylyl-(3'-->5')-thymidine in Escherichia coli under SOS conditions. 867 50

Heavy metals, including zinc (Zn) and cadmium (Cd), are potentially important genotoxic agents in our environment. Here we report that human DNA ligase I, the major form of the enzyme in replicative cells, is a target for Zn and Cd ions. ZnCl2 at 0.8 mM caused complete inhibition of DNA ligase I activity, whereas only 0.04 mM CdCl2 was required to achieve a similar effect. Both metals affected all three steps of the reaction, namely, the formation of ligase-AMP intermediate, the transfer of the AMP to DNA and the ligation reaction that succeeds the formation of the AMP-DNA complex. Unlike F-ara-ATP and the natural protein inhibitor of DNA ligase-I, these metals may affect different domains of the enzyme. Moreover, these metal ions did not increase the rate of misligation of F-ara-A-modified DNA or mismatched DNA substrates, but considerable misligation was observed for the T:C mispairing. These data support the notion of high fidelity of the human DNA ligases and that the major action of these metal ions on the enzyme is their inhibitory function.
Environ Mol Mutagen 1996
PMID:Inhibition of human DNA ligase I activity by zinc and cadmium and the fidelity of ligation. 869 42

In Escherichia coli and related bacteria, the product of gene dcm methylates the second cytosine of 5'-CCWGG sequences (where W is A or T). Deamination of 5-methylcytosine (5meC) results in C to T mutations. The mutagenic potential of 5meC is reduced by a system called very short patch (VSP) repair, which replaces T with C. T:G and U:G mispairs in the methylatable sequence and in related sequences are recognized by the product of vsr, a gene adjacent to dcm. Vsr creates a nick just 5' of the mispaired pyrimidine to initiate the repair. Additional products known to be required for VSP repair are DNA polymerase I and DNA ligase. MutS and MutL have a stimulatory role but are not required. The ability of Vsr to recognize T:G mispairs in sequences related to CCWGG is probably responsible for over- and under-representation of certain tetranucleotides in the E. coli genome. Although VSP repair reduces spontaneous mutations at 5meCs in replicating bacteria, mutation hot-spots persist at these sites. Under conditions that more accurately mimic the natural environment of E. coli, VSP repair appears to be effective in preventing mutation at 5meC.
Mol Microbiol 1996 May
PMID:Very short patch repair: reducing the cost of cytosine methylation. 873 26

Appropriately designed DNA substrates undergo very efficient homologous recombination after injection into the nuclei of Xenopus laevis oocytes. The requirements for this process are that the substrate be linear, that it have direct repeats to support recombination, and that these repeats be at or very near the molecular ends. Taking advantage of direct nuclear injection, the large amounts of DNA processed in a single oocyte, and the accessibility of recombination intermediates, we were able to analyze the mechanism of recombination in detail. Molecular ends are resected by a 5'-->3' exonuclease activity. When complementary sequences are exposed from two ends, they anneal. Continued 5'-->3' degradation removes the redundant strands; the 3' ends pair with their complements and can be extended by DNA polymerase to fill any gap left by the exonuclease. Joining of strands by DNA ligase completes the process. This mechanism is nonconservative, in that only one of the two original repeats is retained, and it has been dubbed single-strand annealing, or SSA. The capability for SSA accumulates during the later phases of oogenesis and persists into the egg. This pattern suggests that, like many activities of full-grown oocytes, SSA is stored for use during embryogenesis. The same or a very similar mechanism is prevalent in many other species, including bacteria, yeast, plants, and mammals, where it often provides the predominant mode of recombination of extrachromosomal DNA. Lessons learned about SSA are applicable to methods of gene manipulation. It is plausible that SSA has a normal function in the repair of double-strand breaks, but proof of this awaits identification of genes and enzymes uniquely involved in this style of recombination.
Prog Nucleic Acid Res Mol Biol 1996
PMID:Homologous genetic recombination in Xenopus: mechanism and implications for gene manipulation. 876 73

Schizosaccharomyces pombe mitochondria were isolated from the cells treated with Novozyme 234, and purified in a Percoll gradient. A zymographic assay in a SDS-polyacrylamide gel containing single-stranded DNA revealed that an endonuclease of 32 kDa is associated with the mitochondria. The endonuclease was extracted from the mitochondria with 0.5 M KCl and was partially purified. The 32-kDa enzyme degraded both DNA and RNA at a weak alkaline pH, but preferred single-stranded DNA. The enzyme required Mg2+ or Mn2+, but not Ca2+ or Zn2+ for activity, and was inhibited by 50% with a 150 mM salt solution. Nicks generated by the enzyme could be resealed with T4 DNA ligase, indicating that the enzyme produces 5'-P and 3'-OH ends.
Biochem Mol Biol Int 1996 Nov
PMID:Identification and characterization of a mitochondrial endonuclease from yeast, Schizosaccharomyces pombe. 895 92

The terminal nucleotide sequence of the Lactobacillus casei bacteriophage A2 DNA revealed a single-stranded extension 13 bases in length (5'-AACGGTCGGCCTC-3') at its 3' termini that defines the packaging initiation nicking site (cosN). The cosN sequence is bisected by an axis of hyphenated twofold rotational symmetry. Directly and inverted repeated sequences located to the left (cosL) and the right (cosR) of the cosN site were observed. Analysis of the 3.4 kb EcoRI DNA sequence surrounding the cos region revealed four complete and one incomplete open reading frames (orfs). Northern blots indicated that all were cotranscribed in a single mRNA molecule in excess of 10 kb that appeared late during infection. Minicell studies indicated that the four orfs were translated into protein. From the ORF3 amino acid sequence DNA-binding and NTP-binding domains can be predicted. The purified ORF3 (predicted molecular mass 16.8 kDa) shows specific binding to the A2 cos region, so it was renamed gp3. Gp3 forms a specific complex with a 369 bp cos DNA segment in the presence of ATP. Gp3 interaction with the intrinsically bent cos DNA segment induces intramolecular ligation in the presence of T4 DNA ligase. The data presented here suggest that gp3 is the small subunit of the terminase enzyme.
Mol Microbiol 1997 Feb
PMID:Molecular analysis of the cos region of the Lactobacillus casei bacteriophage A2. Gene product 3, gp3, specifically binds to its downstream cos region. 904 84

Polymerase chain reaction analysis of a large collection of bacteriophages with T-even morphology revealed four phages that are distantly related to all the others. The genomes of these pseudo T-even phages hybridized under stringent conditions to only a limited portion of the T4 genome that encodes virus head, head-to-tail joining and contractile tail genes. Except for this region, no extensive hybridization was detected between most pairs of the different pseudo T-even genomes. Sequencing of this conserved region of the pseudo T-even phage RB49 revealed substantial nucleotide sequence divergence from T4 (approximately 30% to 40%), and random genomic sequencing of this phage indicated that more than a third of its sequences had no detectable homology to T4. Among those sequences related to the T-even genes were virion structural components including the constituents of the phage base plate. Only a few sequences had homology to T4 early functions; these included ribonucleotide diphosphatase reductase, DNA ligase and the large subunit of DNA topoisomerase. The genomes of the pseudo T-even phage were digested by restriction enzymes that are unable to digest the T-even DNAs which contain glucosylated hydroxymethyl-cytosine residues. This suggests that only limited nucleotide modifications must be present in the pseudo T-even genomes. Conservation of much of the morphogenetic region of these diverse phage genomes may reflect particularly strong sequence constraints on these gene products. However, other explanations are considered, including the possibility that the various morphogenetic segments were acquired by the pseudo T-even genomes by modular evolution. These results support the notion that phage evolution may proceed within a network of both closely and distantly related genomes.
J Mol Biol 1997 Mar 28
PMID:The genome of the pseudo T-even bacteriophages, a diverse group that resembles T4. 909 22

An endonuclease was extracted from intact rat liver mitochondria with 0.4 M NaCl, and partially purified. A zymographic assay in SDS-polyacrylamide gel containing single-stranded DNA revealed that the enzyme has an apparent molecular mass of 55 kDa. It was different from the molecular mass of the major endonuclease of bovine heart mitochondria (a homodimer of a 29-kDa peptide), that was recently shown to be identical to the endonuclease G. The purified 55-kDa enzyme degraded both DNA and RNA, preferring RNA and single-stranded DNA at a weak alkaline pH, required Mg(2+) and Mn(2+) but not Ca(2+) for activity, and was strongly inhibited by monovalent cations. Nicks generated by the enzyme were resealable with T4 DNA ligase, indicating that the enzyme produces 5'-p and 3'-OH ends. The 55-kDa enzyme, like endonuclease G, displayed a strong preference to nick within a (dG)n.(dC)n sequence tract.
Biochem Mol Biol Int 1996 Apr
PMID:Identification of a 55-KDA endonuclease in rat liver mitochondria with nucleolytic properties similar to endonuclease G. 913 52


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