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At least 37 records · Page 2Linked to original sources
PubMed1981

On the cofactor specificity of glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase from chicken liver.

Tests of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and glycinamide ribonucleotide (GAR) transformylase cofactor specificity were conducted with 5-and/or 8-deazafolate analogues formylated at N-10. Several of these compounds were found to serve as cofactors for both the enzymes. The finding that 10-formyl-8-deazafolate can be used by AICAR transformylase eliminates those mechanisms requiring cyclization to a methenyl derivative prior to carbon unit transfer for this transformylase. Surprisingly, a similar analogue, 10-formyl-5,8-deazafolate, is very effective as a cofactor for GAR transformylase in the presence or absence of the trifunctional protein which is required for 5,10-methenyl-H4-folate activity with this transformylase. This finding suggests that the trifunctional protein modulates GAR transformylase cofactor specificity by supplying the active cofactor as the N10-formyl species, possibly through a transport process that avoids its dissociation into solution.

Acyltransferases↗
PubMed1990

Cloning of a chicken liver cDNA encoding 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase by functional complementation of Escherichia coli pur mutants.

We have used functional complementation of Escherichia coli pur mutants to clone avian cDNA encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase-5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase, the bifunctional enzyme catalyzing steps 6 and 7 in the pathway for de novo purine nucleotide synthesis. Mutational analyses have been used to establish the structure-function relationship: NH2-SAICAR synthetase-AIR carboxylase-COOH. The amino acid sequence of the SAICAR synthetase domain is homologous to that of bacterial purC-encoded enzymes, and the sequence of the following AIR carboxylase domain is homologous to that of bacterial purE-encoded enzymes. In E. coli, AIR carboxylase is the product of genes purEK with the purK subunit postulated to have a role in CO2 binding. The avian enzyme lacks sequences corresponding to purK yet functions in E. coli. Functional complementation of E. coli pur mutants can be used to clone additional avian cDNAs for de novo purine nucleotide synthesis.

Amino Acid Sequence↗
PubMed2003

Ribonucleotide reduction in Mycobacterium tuberculosis: function and expression of genes encoding class Ib and class II ribonucleotide reductases.

Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the nrdE and nrdF2 genes, in addition to a putative class II RNR, encoded by nrdZ. In this study we probed the relative contributions of these RNRs to the growth and persistence of M. tuberculosis. We found that targeted knockout of the nrdF2 gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the nrdF1 gene is unable to substitute for nrdF2 and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a DeltanrdZ null mutant of M. tuberculosis was readily obtained by allelic exchange mutagenesis. Quantification of levels of nrdE, nrdF2, nrdF1, and nrdZ gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O(2)-limited cultures showed that nrdZ was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O(2) restriction. However, survival of the DeltanrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F(1) mice infected with the DeltanrdZ mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that nrdZ plays a significant role in the virulence of M. tuberculosis in this mouse model.

Animals↗
PubMed1981

Effects of altered ribonucleotide concentrations on ribonucleotide reduction in intact Chinese hamster ovary cells.

The effect of changes in ribonucleoside triphosphate concentrations on those of deoxyribonucleotides has been determined in Chinese hamster ovary K-1 cells. Ribonucleoside triphosphate concentrations were manipulated by the use of drugs or naturally occurring purines or pyrimidines. Changes in the concentration of each ribonucleoside triphosphate led to changes in the concentration of each corresponding deoxyribonucleoside triphosphate. The relationships between the concentrations of CTP and dCTP and between those of GTP and dGTP were linear over a wide range. The relationship between the concentrations of ATP and dATP was linear over the narrow range studied, while the relationship between UTP and dTTP concentrations was curvilinear. Overall, these results demonstrate that ribonucleotides play an important role in controlling deoxyribonucleotide concentrations but the exact mechanism of this control remains to be determined.

Animals↗
PubMed1995

[Glycine amide ribonucleotide synthetase (EC 6.3.4.13)--is aminoimidazole ribonucleotide synthetase (EC 6.3.3.1) from Saccharomyces cerevisiae].

The bifunctional enzyme GAR-synthetase-AIR-synthetase (E2-E5) of the yeast Saccharomyces cerevisiae has been studied. The yeast strain with overproduction of E2-E5 has been obtained. The enzyme from this strain, E2-E5, has been purified and characterized. The protein is a dimer composed of two subunits with M(r) of 87 kDa. The pH and temperature optima, pH stability and thermostability for E2 and E5 have been determined. The kinetic constants for E2 and E5 have been estimated. E2 and E5 are active only in the presence of Mg2+. E5 is a K(+)-dependent enzyme as is E5 from other sources. AMP is a competitive (to ATP) inhibitor for E5; hence, in yeast cells the purine nucleotide biosynthesis de novo is regulated at the first and fifth steps. Partial chymotryptic digestion of the purified protein gives rise to two fragments with M(r) of about 40 and 46 kDa; and E2 activity remains, while that of E5 disappears in the process.

Amino Acid Sequence↗
PubMed2026

Fluorescent reporter assay reveals ribonucleotides promote mismatch correction in vivo.

Ribonucleotides can serve as a strand discrimination signal in reconstituted in vitro biochemical mismatch repair (MMR) assays, but the influence of ribonucleotides on mismatch correction has not been measured directly in vivo. We have developed a fluorescence-based host cell reactivation assay that reports correction of a mismatch in proximity of a site-specifically incorporated ribonucleotide. A ribonucleotide leads to enhanced mismatch correction. While neither inactivation of a single allele nor knockdown of RNaseH2 is sufficient to suppress ribonucleotide directed MMR, a modest but statistically significant impairment for repair of mismatches in the presence of an embedded ribonucleotide is observed in RNaseH2 knockout cell lines. Reporter plasmids with ribonucleotides located in either the 3' or 5' orientation are robustly repaired in MMR-proficient cells but are weakly repaired in MMR-deficient cells, underscoring their utility as effective MMR reporters. Significant ribonucleotide-enhanced mismatch correction was consistently observed in MMR-deficient cells when the ribonucleotide is in the 3' orientation. The presence of a ribonucleotide led to enhanced MMR even in RNaseH2 knockout cells, suggesting that other enzymes may promote ribonucleotide-directed MMR. Loss of RNaseH2 was not sufficient to confer significant resistance to the alkylating agent, temozolomide, in support of a model in which ribonucleotide-directed repair events make minor contributions to the canonical MMR pathway in mammalian cells. We propose a model in which MMR-independent ribonucleotide enhanced correction of mismatches can proceed by ribonucleotide excision repair when the ribonucleotide is in the 5' direction, and proceeds by an unknown mechanism when the ribonucleotide is in the 3' direction.

DNA Mismatch Repair↗
PubMed2000

Position-specific effect of ribonucleotides on the cleavage activity of human topoisomerase II.

Beyond the normal DNA transactions mediated by topoisomerase II, we have recently demonstrated that the cleavage activity of the two human topoisomerase II isoforms is several-fold stimulated if a ribonucleotide rather than a deoxyribonucleotide is present at the scissile phosphodiester in one strand of the substrate. Here we show that ribonucleotides exert a position-specific effect on topoisomerase II-mediated cleavage without altering the sequence specificity of the enzyme. Ribonucleotides located within the 4 bp cleavage stagger stimulate topoisomerase II-mediated cleavage, whereas ribonucleotides located outside the stagger in general have an inhibitory effect. Results obtained from competition experiments indicate that the position-specific effect of ribonucleotides on topoisomerase II activity is caused by altered substrate interaction. When cleavage is performed with substrates containing one ribonucleotide in both strands or several ribonucleotides in one strand the effect of the individual ribonucleotides on cleavage is not additive. Finally, although topoisomerase II recognizes substrates with longer stretches of ribonucleotides, an RNA/DNA hybrid where one strand is composed entirely of RNA is not cleaved by the enzyme. The positional effect of ribonucleotides on topoisomerase II-mediated cleavage shares many similarities to the positional effect exerted by either abasic sites or base mismatches, demonstrating a general influence of DNA imperfections on topoisomerase II activity.

Base Pair Mismatch↗