Thioredoxin-C': mechanism of noncovalent complementation and reactions of the refolded complex and the active site containing fragment with thioredoxin reductase

Holmgren, Arne; Slaby, Ivan

doi:10.1021/bi00592a011

Cited by 28 publications

(20 citation statements)

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“…The viability of E. coli mutants lacking thioredoxin or thioredoxin reductase indicated that alternative pathways for ribonucleotide reduction existed and led to the discovery of glutaredoxins (24). In vitro ribonucleotide reductase assays showed that glutaredoxin was almost 10-fold more active than thioredoxin on a molar basis (25). However, thioredoxin is 10-fold more abundant than glutaredoxin in the E. coli cytosol (25).…”

Section: Discussionmentioning

confidence: 99%

“…In vitro ribonucleotide reductase assays showed that glutaredoxin was almost 10-fold more active than thioredoxin on a molar basis (25). However, thioredoxin is 10-fold more abundant than glutaredoxin in the E. coli cytosol (25). An extract from an E. coli mutant lacking thioredoxin showed high levels of NADPH-dependent ribonucleotide reductase activity (26).…”

Section: Discussionmentioning

confidence: 99%

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Thioredoxin Is Required for Deoxyribonucleotide Pool Maintenance during S Phase

Koç¹,

Mathews

Wheeler

et al. 2006

Journal of Biological Chemistry

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Thioredoxin was initially identified by its ability to serve as an electron donor for ribonucleotide reductase in vitro. Whether it serves a similar function in vivo is unclear. In Saccharomyces cerevisiae, it was previously shown that ⌬trx1 ⌬trx2 mutants lacking the two genes for cytosolic thioredoxin have a slower growth rate because of a longer S phase, but the basis for S phase elongation was not identified. The hypothesis that S phase protraction was due to inefficient dNTP synthesis was investigated by measuring dNTP levels in asynchronous and synchronized wild-type and ⌬trx1 ⌬trx2 yeast. In contrast to wild-type cells, ⌬trx1 ⌬trx2 cells were unable to accumulate or maintain high levels of dNTPs when ␣-factor-or cdc15-arrested cells were allowed to reenter the cell cycle. At 80 min after release, when the fraction of cells in S phase was maximal, the dNTP pools in ⌬trx1 ⌬trx2 cells were 60% that of wild-type cells. The data suggest that, in the absence of thioredoxin, cells cannot support the high rate of dNTP synthesis required for efficient DNA synthesis during S phase. The results constitute in vivo evidence for thioredoxin being a physiologically relevant electron donor for ribonucleotide reductase during DNA precursor synthesis.Deoxyribonucleotide pools are carefully controlled in all cells to ensure efficient and yet accurate genome replication. The pools are not equimolar, but rather they have a characteristic asymmetry, with dGTP usually the smallest (1). For example, in Saccharomyces cerevisiae, the dGTP pool is 3-fold smaller than the dTTP pool (2, 3). Cells do not stockpile dNTPs, but rather they maintain them at levels just sufficient to support replication. For example, yeast contain fewer than 1.8 ϫ 10 6 dNTP molecules/cell (3), which is less than 20% of the amount minimally needed to copy the 1.2 ϫ 10 7 -bp yeast genome. Limiting dNTP pool size is important for replication fidelity. Artificial pool expansion results in increased mutagenesis, perhaps because of stimulation of chain extension from DNA mismatches at high dNTP concentrations (4). The dNTP pools are dynamic, increasing severalfold as eukaryotic cells enter S phase (3). For dNTP levels to remain elevated during S phase, cells must markedly boost their rate of dNTP synthesis to compensate for the rapid consumption of dNTPs at replication forks. Replicating cells are exquisitely sensitive to dNTP depletion. When dNTP synthesis is blocked by the addition of hydroxyurea, replication stops well before any dNTP pool is exhausted (5). Presumably, cells have evolved the mechanisms to limit dNTP accumulation to support replication fidelity and yet maintain dNTP levels above the minima required for efficient DNA replication and repair.A central enzyme controlling the rate of dNTP synthesis is ribonucleotide reductase (RNR), 3 a heterotetramer of two large and two small subunits. The enzyme is subject to extraordinary regulation. Its activity and substrate specificity are regulated by binding of different nucleoside triphosphates to two allo...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Thioredoxin Is Required for Deoxyribonucleotide Pool Maintenance during S Phase

Koç¹,

Mathews

Wheeler

et al. 2006

Journal of Biological Chemistry

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“…Proteolytic or recombinant fragments of a number of enzymes are known to reassociate in vitro to reconstitute activity (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48). In addition, a systematic analysis of functional assembly of a large aminoacyl-tRNA synthetase (939 residues) from fragment pairs and triplexes has been undertaken (49)(50)(51) (10).…”

Section: Discussionmentioning

confidence: 99%

Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments.

Severinov

Mustaev

Severinova

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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The Escherichia coli rpoB gene, which codes for the 1342-residue j3 subunit of RNA polymerase (RNAP), contains two dispensable regions centered around codons 300 and 1000. To (26). In this report we extend these studies andThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.show that the 3 subunit split at dispensable region I and at the two dispensable regions simultaneously can still assemble into a functional enzyme in vitro. These results indicate that the 3 subunit is composed of at least three independent domains and delineate a strategy that can be used to define further the domain architecture of the RNAP subunits. MATERIALS AND METHODSGenetic Techniques. The pUC19-based pMKA92 rpoB expression plasmid has been described (27

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“…Limited proteolytic cleavage of ribonuclease A, staphylococcal nuclease, cytochrome c, cytochrome b5 reductase, human pituitary growth hormone, human hemoglobin, thioredoxin C, adenylate kinase, or alanine racemase yields fragments that can reassociate in vitro to reconstitute activity (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). These protease-generated fragments are believed to represent well-defined structural units or domains that associate through complementary sequences at the interfaces.…”

mentioning

confidence: 99%

Functional assembly of a randomly cleaved protein.

Shiba

Schimmel

1992

Proc. Natl. Acad. Sci. U.S.A.

104

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The sequence of a 939-amino acid polypeptide that is a member of the aminoacyl-tRNA synthetase class of enzymes has been aligned with sequences of 15 related proteins. This alignment guided the design of 18 fragment pairs that were tested for internal sequence complementarity by reconstitution of enzyme activity. Reconstitution was achieved with fragments that divide the protein at both nonconserved and conserved sequences, including locations proximal to or within elements believed to form critical elements of secondary structure. Structure assembly is sufficiently flexible to accommodate fusion of short segments of unrelated sequences at fragment junctions. Complementary chain packing interactions and chain flexibility appear to be widely distributed throughout the sequence and are sufficient to reconstruct large threedimensional structures from an array of disconnected pieces. The results may have implications for the evolution and assembly of large proteins.

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Thioredoxin-C': mechanism of noncovalent complementation and reactions of the refolded complex and the active site containing fragment with thioredoxin reductase

Cited by 28 publications

References 20 publications

Thioredoxin Is Required for Deoxyribonucleotide Pool Maintenance during S Phase

Thioredoxin Is Required for Deoxyribonucleotide Pool Maintenance during S Phase

Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments.

Functional assembly of a randomly cleaved protein.

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