Graduate Thesis Or Dissertation

 

Cell cycle regulation of DNA precursor accumulation in the yeast Saccharomyces cerevisiae Public Deposited

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  • In budding yeast, many of the genes that encode enzymes required for DNA precursor synthesis (MCB genes) are expressed under cell cycle control in late G1/S. The relationship between MCB gene expression, dNTP synthesis and DNA synthesis was investigated by using α factor-synchronized Saccharomyces cerevisiae. The levels of all four dNTPs increased several-fold when cells crossed the G1/S boundary. An even larger increase in the dNTP pools occurred at G1/S when replication initiation was blocked by incubating synchronized dbf4 mutants at the nonpermissive temperature. Thus, dNTP accumulation at G1/S was not dependent on replication initiation. Similarly, MCB gene induction at G1/S was also independent of replication initiation. The accumulation of dNTPs at G1/S was dependent on Swi6, a protein known to be required for normal MCB gene regulation during the cell cycle. Treatment with hydroxyurea, an inhibitor of ribonucleotide reductase, blocked DNA synthesis and prevented the increase in dNTP levels that normally occurred at G1/S, however, it did not exhaust the basal levels of any of the four dNTPs. The mechanism responsible for replication arrest despite the persistence of dNTPs was not dependent on the checkpoint protein Rad53, as rad53 mutants also failed to exhaust basal dNTPs when incubated in HU. The inhibitory effect of HU on DNA synthesis was bypassed when dbf4 cells were allowed to pre-accumulate dNTPs at 37°C before being released to the permissive temperature in the presence of HU. Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, as dbf4 cells incubated in HU at 25°C were able to initiate replication when cells were switched to the nonpermissive temperature and HU was removed. The results indicate that DNA chain elongation in yeast requires a critical dNTP threshold, below which replication forks are completely arrested. Cells lacking a functional thioredoxin system were deficient in dNTP synthesis. The rate of accumulation was significantly lower in Δtrr1 mutants lacking thioredoxin reductase, and dNTP accumulation at G1/S did not occur at all in Δtrxl Δtrx2 double mutants lacking thioredoxin. The results suggest that thioredoxin serves as the electron donor for ribonucleotide reductase during DNA precursor synthesis in yeast.
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