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    Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools
    (J Biol Chem, 2003) Koç, Ahmet; Wheeler, Linda J.; Mathews, Christopher K.
    The relationship between dNTP levels and DNA synthesis was investigated using factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G1/S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37 °C before being released to 25 °C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G1 levels. Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25 °C were able to replicate when subsequently switched to 37 °C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/ Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold.
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    Replication independent MCB gene induction and deoxyribonucleotide accumulation at G1 S in Saccharomyces cerevisiae
    (J Biol Chem, 2003) Koç, Ahmet; Wheeler, Linda J.; Mathews, Christopher K.; Merrill, Gary F.
    In Saccharomyces cerevisiae, many genes encoding enzymes involved in deoxyribonucleotide synthesis are expressed preferentially near the G1/S boundary of the cell cycle. The relationship between the induction of deoxyribonucleotide-synthesizing genes, deoxyribonucleoside triphosphate levels, and replication initiation was investigated using factor-synchronized wildtype yeast or dbf4 yeast that are temperature-sensitive for replication initiation. Neither the timing nor extent of gene induction was inhibited when factor-arrested dbf4 cells were released into medium containing the ribonucleotide reductase inhibitor hydroxyurea, which blocks replication fork progression, or were released at 37 °C, which blocks replication origin firing. Thus, the induction of deoxyribonucleotide-synthesizing genes at G1/S was fully independent of DNA chain elongation or initiation. Deoxyribonucleoside triphosphate levels increased severalfold at G1/S in wild-type cells and in dbf4 mutants incubated at the non-permissive temperature. Thus, deoxyribonucleoside triphosphate accumulation, like the induction of deoxyribonucleotide-synthesizing genes, was not dependent on replication initiation. Deoxyribonucleoside triphosphate accumulation at G1/S was suppressed in cells lacking Swi6, a transcription factor required for normal cell cycle regulation of deoxyribonucleotide-synthesizing genes. The results suggest that cells use gene induction at G1/S as a mechanism to pre-emptively, rather than reflexively, increase the synthesis of DNA precursors to meet the demand of the replication forks for deoxyribonucleotides.
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    Thioredoxin is required for deoxyribonucleotide pool maintenance during S phase
    (J Biol Chem, 2006) Koç, Ahmet; Mathews, Christopher K.; Wheeler, Linda J.; Gross, Michael K.; Merrill, Gary F.
    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.