Thioredoxin Is Required for Deoxyribonucleotide Pool Maintenance during S Phase

Abstract

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...

Checkpoint deficient rad53-11 yeast cannot accumulate dNTPs in response to DNA damage

“…Although the DNA damage response and dNTP synthesis pathways of yeast and mammals have significant similarities, it is neither clear why mammalian cells do not show an increase in the sizes of their dNTP pools nor how they perform the repair process in the event of DNA damage. Our dNTP pool measurements in wild-type and rad53-11 mutant cells were consistent with previously published results [7,[13][14][15]. In one of our previous studies, similar dNTP levels were observed for asynchronous rad53-11 cells, and mutants did not show any cell-cycle perturbations when analysed by flow cytometry, which suggested that rad53-11 cells operate under a normal cell-cycle progression.…”

Gemcitabine chemoresistance and molecular markers associated with gemcitabine transport and metabolism in human pancreatic cancer cells

“…It has recently been reported that the physiological factor for ribonucleotide reduction is thioredoxin, which is required for the RR reaction in vitro (Koc et al, 2006). In yeast, mutants lacking thioredoxin had significantly lower dNTP levels, supporting the idea that thioredoxin functions as an RR reductant in vitro.…”

Determination of the in Vivo Stoichiometry of Tyrosyl Radical per ββ‘ in Saccharomyces cerevisiae Ribonucleotide Reductase

“…In all eukaryotic systems that were examined, with the exception of our studies, DTT is used as the reductant to allow RNR to catalyze multiple turnovers, rather than the endogenous reductant, thioredoxin and thioredoxin reductase (76). In our hands when α is assayed with excess ββ′, the activity is reduced several-fold with DTT in place of the protein reducing system (data not shown).…”

“…From our data, and the range of specific activities for α n and ββ′, the number of dNTPs produced can be calculated to vary between 3.6 × 10 8 and 1.1 × 10 9 , a 10-100-fold excess over the requirements for genome replication. Recent dNTP pool measurements by Merrill and co-workers indicate that in asynchronous wt S. cerevisiae cells (W303) there are 1.8 × 10 6 dNTPs, only 10% of the pool size minimally required to replicate the whole genome (76). Thus, the low levels of dNTPs measured by Koc et al suggest that RNR activity is greatly reduced in vivo.…”