Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase. Enhancement of adenosine N1-oxide reductase activity.
(9/3529)
The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrificans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R. sphaeroides or E. coli N-terminal signal sequence. Whereas the R. sphaeroides signal sequence prevents formation of active enzyme, addition of a 6x His-tag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification. The recombinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum atoms per molecule and has a DMSO reductase activity of 3.4-3.7 units/nmol molybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R. sphaeroides. The recombinant enzyme differs from the native enzyme in its color and spectrum but is indistinguishable from the native protein after redox cycling with reduced methyl viologen and Me2SO. Substitution of Cys for the molybdenum-ligating Ser-147 produced a protein with DMSO reductase activity of 1.4-1.5 units/nmol molybdenum. The mutant protein differs from wild type in its color and absorption spectrum in both the oxidized and reduced states. This substitution leads to losses of 61-99% of activity toward five substrates, but the adenosine N1-oxide reductase activity increases by over 400%. (+info)
A cytotoxic ribonuclease targeting specific transfer RNA anticodons.
(10/3529)
The carboxyl-terminal domain of colicin E5 was shown to inhibit protein synthesis of Escherichia coli. Its target, as revealed through in vivo and in vitro experiments, was not ribosomes as in the case of E3, but the transfer RNAs (tRNAs) for Tyr, His, Asn, and Asp, which contain a modified base, queuine, at the wobble position of each anticodon. The E5 carboxyl-terminal domain hydrolyzed these tRNAs just on the 3' side of this nucleotide. Tight correlation was observed between the toxicity of E5 and the cleavage of intracellular tRNAs of this group, implying that these tRNAs are the primary targets of colicin E5. (+info)
Msh2 status modulates both apoptosis and mutation frequency in the murine small intestine.
(11/3529)
Deficiency in genes involved in DNA mismatch repair increases susceptibility to cancer, particularly of the colorectal epithelium. Using Msh2 null mice, we demonstrate that this genetic defect renders normal intestinal epithelial cells susceptible to mutation in vivo at the Dlb-1 locus. Compared with wild-type mice, Msh2-deficient animals had higher basal levels of mutation and were more sensitive to the mutagenic effects of temozolomide. Experiments using Msh2-deficient cells in vitro suggest that an element of this effect is attributable to increased clonogenicity. Indeed, we show that Msh2 plays a role in the in vivo initiation of apoptosis after treatment with temozolomide, N-methyl-N'-nitro-N-nitrosoguanidine, and cisplatin. This was not influenced by the in vivo depletion of O6-alkylguanine-DNA-alkyltransferase after administration of O6-benzylguanine. By analyzing mice mutant for both Msh2 and p53, we found that the Msh2-dependent apoptotic response was primarily mediated through a p53-dependent pathway. Msh2 also was required to signal delayed p53-independent death. Taken together, these studies characterize an in vivo Msh2-dependent apoptotic response to methylating agents and raise the possibility that Msh2 deficiency may predispose to malignancy not only through failed repair of mismatch DNA lesions but also through the failure to engage apoptosis. (+info)
Leukocyte O6-alkylguanine-DNA alkyltransferase from human donors is uniformly sensitive to O6-benzylguanine.
(12/3529)
O6-Alkylguanine-DNA alkyltransferase (AGT) is the key DNA repair protein responsible for resistance to chloroethylating and methylating agents that attack at the O6 position of guanine. O6-Benzylguanine (BG), a potent inhibitor of AGT, has recently entered clinical trials. A number of point mutations and at least one human polymorphism within AGT are associated with AGT resistance to inactivation by BG. In this study, we evaluated AGT inhibition by BG in an in vitro assay of peripheral blood mononuclear cell AGT from 56 normal donors, 42 Caucasians, and 14 Japanese. AGT activity ranged from 2.7 to 21.9 fmol/microg DNA and was similar in Japanese and Caucasian donors. Depletion of AGT by BG was uniform in all donors with mean ED50s of 037 microM BG in Caucasians and 0.36 microM BG in Japanese. To determine whether the gly160arg AGT polymorphism described in the Japanese population, and recently shown to be BG resistant, could be detected by this assay, we mixed purified gly160arg AGT protein with blood mononuclear cell extract and measured in vitro BG inactivation. The ED50 for the mixture of the gly160arg AGT and mononuclear cell extract was 9 microM BG. On the basis of results in 56 donors, we conclude that BG-resistant AGT, defined as an ED50 in mononuclear cells of >1 microM BG, is present in 0 of 56 donors, (95% confidence interval, 0-6%), suggesting that polymorphisms producing AGT-resistant BG are unusual in humans. (+info)
Barminomycin forms GC-specific adducts and virtual interstrand crosslinks with DNA.
(13/3529)
The sequence specificity of the binding of barminomycin (SN-07 chromophore) to DNA was investigated using an in vitro transcription assay. It was found that this compound formed blockages to transcription, and these blocks were highly selective for 5'-GC sequences. The half-lives of the first seven transcriptional blockages at 37 degrees C were 14-130 min, plus one site >>200 min, with widely varying levels of essentially permanent blockages at each site (0-100%; average of 40%), indicative of considerable dependence on flanking sequences of adducts stability at individual GC sites. Barminomycin was also shown to form DNA virtual (i.e. functional) interstrand crosslinks. Such crosslinks were also relatively heat stable, with 40% of the DNA remaining crosslinked after heating at 90 degrees C for 5 min. The barminomycin-DNA adducts and crosslinks appear to be essentially identical to those formed between adriamycin and DNA. Whereas adriamycin requires prior activation with formaldehyde in order to form adducts and crosslinks, barminomycin behaves in all respects as if it is a pre-activated form of adriamycin. (+info)
Identification and characterisation of the Drosophila melanogaster O6-alkylguanine-DNA alkyltransferase cDNA.
(14/3529)
The protein O 6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved in the repair of O 6-alkylguanine and O 4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents. A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene ( DmAGT ) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man. The DmAGT gene is located at position 84A on the third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA. Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O 6-benzylguanine. In an E.coli lac Z reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-->A:T as well as A:T-->G:C transition mutations in vivo. These results demonstrate the presence of an alkyltransferase specific for the repair of O 6-methylguanine and O 4-methylthymine in Drosophila. (+info)
Reactivity of potassium permanganate and tetraethylammonium chloride with mismatched bases and a simple mutation detection protocol.
(15/3529)
Many mutation detection techniques rely upon recognition of mismatched base pairs in DNA hetero-duplexes. Potassium permanganate in combination with tetraethylammonium chloride (TEAC) is capable of chemically modifying mismatched thymidine residues. The DNA strand can then be cleaved at that point by treatment with piperidine. The reactivity of potassium permanganate (KMnO4) in TEAC toward mismatches was investigated in 29 different mutations, representing 58 mismatched base pairs and 116 mismatched bases. All mismatched thymidine residues were modified by KMnO4/TEAC with the majority of these showing strong reactivity. KMnO4/TEAC was also able to modify many mismatched guanosine and cytidine residues, as well as matched guanosine, cytidine and thymidine residues adjacent to, or nearby, mismatched base pairs. Previous techniques using osmium tetroxide (OsO4) to modify mismatched thymidine residues have been limited by the apparent lack of reactivity of a third of all T/G mismatches. KMnO4/TEAC showed no such phenomenon. In this series, all 29 mutations were detected by KMnO4/TEAC treatment. The latest development of the Single Tube Chemical Cleavage of Mismatch Method detects both thymidine and cytidine mismatches by KMnO4/TEAC and hydroxylamine (NH2OH) in a single tube without a clean-up step in between the two reactions. This technique saves time and material without disrupting the sensitivity and efficiency of either reaction. (+info)
Synthesis and salvage of purines during cellular morphogenesis of Myxococcus xanthus.
(16/3529)
Intact cells of Myxococcus xanthus were examined for de novo purine synthesis and salvage utilization. The cellular uptake rates of radioactive glycine (de novo purine precursor), adenine, and guanine were measured, and thin-layer chromatography and radioautography were used to examine cell extracts for de novo synthesized purine nucleotides. Intact vegatative cells, glycerol-induced myxospores, and germinating cells of M. xanthus CW-1 were able to carry out de novo purine and salvage synthesis. Germinating cells and glycerol-induced myxospores were metabolically more active or as active as vegetative cells with respect to purine anabolism. We conclude that M. xanthus is capable of synthesizing purine nucleotides and salvaging purines throughout the glycerol version of its life cycle. (+info)