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 �� 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 ��trr1
mutants lacking thioredoxin reductase, and dNTP accumulation at G1/S did not
occur at all in ��trxl ��trx2 double mutants lacking thioredoxin. The results suggest
that thioredoxin serves as the electron donor for ribonucleotide reductase during
DNA precursor synthesis in yeast. / Graduation date: 2003
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/32555 |
| Date | 11 June 2002 |
| Creators | Koc, Ahmet |
| Contributors | Merrill, Gary F. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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