Recombination and hydroxyurea inhibition of DNA synthesis in yeast meiosis

Abstract

Hydroxyurea (HU) inhibits the premeiotic DNA replication and the meiotic events that followed, namely readiness, recombination commitment, haploidisation, sporulation commitment and ascus formation. Short incubations with HU (2-4 hrs) during the premeiotic replication (i.e. starting between 3 and 6.5 hrs in sporulation medium) allow the resumption of the replication at a normal rate following the removal of the drug. The other meiotic events are similarly delayed by the approximate length of the treatment. In these experiments, intragenic recombination in ade2 reached a higher level than in the controls (x 1.3-2.0 in one pair of heteroalleles and x 3.0-4.0 in another pair). The recombination response to short HU treatments was not observed for a pair of heteroalleles in ade2 that normally shows a high level of meiotic recombination (750 per 10(6) cells), nor was the response observed in a pair of heteroalleles in lys2. HU treatments have almost no effect on sporulating cells from 8 hrs onwards. At 7-7.5 hrs the meiotic cells are very sensitive to the drug and even short treatments cause cell death and massive DNA degradation.

CLB5 and CLB6 are required for premeiotic DNA replication and activation of the meiotic S/M checkpoint

“…clb5 clb6 mutants proceed through the meiotic program in the absence of replicated DNA and undergo a catastrophic M phase Chemical inhibitors or mutations that block premeiotic DNA replication also block progression into the meiotic program, decrease or prevent meiotic recombination, and inhibit the expression of middle and late sporulation genes (Simchen et al 1976;Schild and Byers 1978;Mitchell 1994). Thus, the failure to replicate chromosomal DNA would alone be sufficient to explain the inability of clb5/clb5 clb6/clb6 mutants to complete meiosis and undergo sporulation, as indicated by the sporulation defect observed in HU-treated cells (Table 1).…”

“…Stable arrest in response to inhibitors and mutations that block DNA replication during meiosis is well documented (Simchen et al 1976;Schild and Byers 1978). This response can now be attributed to the existence of a MEC1-dependent checkpoint that monitors DNA replication and blocks meiotic progression.…”

“…Many of the same gene products required for DNA replication during the mitotic cycle are also essential for meiosis. These include enzymes involved in the synthesis of DNA precursors (TMP1, RNR1, and CDC8) and in the process of replication itself (CDC2, CDC17, and CDC9) (Simchen et al 1976;Zamb and Roth 1977;Schild and Byers 1978;Johnson et al 1982;Budd et al 1989). In addition, it has been established that the same replication origins are used in both meiotic and mitotic S phases (Collins and Newlon 1994).…”

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

“…3F). To test whether the appearance of the high fluorescence peak requires DNA replication, strains X488-2 POLl and X488-17-2 poll-17 were sporulated at 23°C in the presence of hydroxyurea, which has been shown in several studies to be an inhibitor of elongation during premeiotic DNA synthesis (36,39). Figure 3G and H illustrates that the appearance of the high fluorescence peak is significantly reduced in the presence of hydroxyurea.…”

THE TEMPORAL RESPONSE OF RECOMBINATION EVENTS TO GAMMA RADIATION OF MEIOTIC CELLS INSORDARIA BREVICOLLIS

“…Both diploid and disomic systems have been used to demonstrate that neither intergenic nor intragenic recombination is possible without premeiotic DNA synthesis. This has been shown with hydroxyurea inhibition of DNA synthesis (Silva-Lopez et al, 1975;Simchen et al, 1976) and by the use of temperature sensitive mutations which block premeiotic DNA replication (Simchen and Hirshberg, 1977). Similarly in Chlamydomonas inhibitors of premeiotic DNA synthesis also reduce recombination frequency (Chiu and Hastings, 1973).…”