Our studies point to Cdc28p activity being tightly regulated during meiotic S phase in order to ensure that only one round of meiotic DNA replication is initiated. One level of regulation requires Swe1p, as diploid cells lacking this kinase activity form multispore asci. Presumably, swe1 cells contain deregulated and possibly hyperactivated Cdc28p activity that lacks Y19 inhibitory phosphorylation. Moreover, our results further support the idea that Cdc28p activity plays a primary role in meiotic replication, most likely through a Cdc28p-Clb5p complex.
We found that swe1
cells lacking Cdc28p-Clb5p activity did not form multispore asci or initiate meiotic DNA replication, while cells lacking Clb6p were multispore competent. However, cells lacking Clb5p did not even initiate meiotic replication in our strain background. Thus, our results can suggest only that the execution point of Clb5p-Cdk activity and meiotic rereplication initiation are epistatic, as is Cdc55p-dependent PP2A activity. Strich et al. (45
) recently demonstrated that constitutive overexpression of CLB1 during meiosis causes multispore formation. swe1
cells do induce CLB1 earlier and temporally sustain its expression level longer than seen in wild-type cells (Fig. ). Whether sustained levels of CLB1
play a role in the multispore phenotype of swe1
cells is currently being pursued.
Yeast strains recalcitrant to Cdc28p-dependent regulation of ORC, Mcm2-7p, and Cdc6p together are defective in blocking mitotic replication reinitiation (38
). However, not all origins reinitiate replication in this triply deregulated strain, suggesting that additional regulatory mechanisms exist and help to prevent unscheduled DNA replication. By deregulating Cdc28p activity, we were able to permit a subset of cells to reinitiate meiotic replication. Possibly the additional mechanism alluded to functions through directly regulating Cdc28p activity. Another possibility is that meiotic replication initiation may be under less stringent control, since only a single S phase is initiated prior to the initiation of a terminal ascus phenotype. Therefore, loss of Cdc28p regulation alone can give rise to reinitiation of meiotic, but not mitotic, replication. We need to explore the “activity status” of ORC, Mcm2p-7p, and Cdc6p in Cdc28p deregulated strains in order to better understand how deregulation of Cdc28p leads to replication reinitiation during meiosis.
We would like to examine whether deregulated Cdc28p either inhibits activities necessary to set up a meiotic post-RC or simply keeps the already formed pre-RC in a functional state. Liang and Stillman (28
) have shown that cells harboring cdc6-3
alone are able to reinitiate mitotic replication. This allele expresses a Cdc6p that is stable and resistant to Cdc28p-dependent negative regulation. Also it has been shown that high-level expression of the Schizosaccharomyces pombe
ortholog Cdc18 induces multiple rounds of DNA replication (37
). Cdc6p is degraded through the SCF/Cdc34p ubiquitin-ligase complex (51
). Rudner et al. (42
) have evidence showing that cells harboring a CDC28
allele nearly identical to CDC28AF
have reduced activity of the anaphase-promoting ubiquitin-ligase complex, delaying exit from mitosis. We are currently examining whether Cdc28p has a role in regulating SCF activity during meiosis, or if mutant cells harbor reduced anaphase-promoting complex activity, and whether either contributes to multispore formation.
Why is initiating homologous recombination essential for multispore formation in cells deregulated for Cdc28p activity? The meiotic cell may consider homologous recombination part of meiotic S phase; thus, the post-RC resistant to reinitiation of replication (19
) is generated only after recombination has been completed. An active pre-RC therefore remains bound to origins during recombination, and the deregulation of Cdc28p becomes the driving force for multiple rounds of replication. We ruled out the possibility that untimely transient activation of either the DNA damage (32
) or pachytene (40
) checkpoint during recombination, which could possibly cause the down-regulation of Cdc28p activity and subsequent reformation of a competent pre-RC, was responsible for multispore formation.
Spo11p has a function that is independent of its role in DSB initiation. It, along with Rec8p, is involved in determining the length of meiotic S phase; loss of Spo11p during meiosis shortens meiotic S phase, whereas loss of Rec8p causes an increase (4
). It is thought that Spo11p may directly integrate the formation of interhomolog and intersister interactions with ongoing meiotic replication (4
), either through a direct interaction with chromatin or through its possibly interacting with specific replication factors (12
). Thus, we cannot rule out that the role of Spo11p in initiating meiotic rereplication is to form these stable chromosomal intermediates or interact with replication initiators, rather than initiating DSBs. The fact that loss of REC102
abolishes multispore formation does lend support to the idea that initiating homologous recombination is necessary.
We found that Spo13p was required for rereplication and multispore formation. Interestingly, Spo13p has a role in protecting Rec8p from cleavage by the separase Esp1p (24
diploid cells have lower levels of Rec8p on centromeres during anaphase of meiosis I (23
). Forsburg (12
) has suggested that, during meiotic S phase, cells may assemble a “prerecombination complex” that contains Spo11p, with the activity of this complex possibly being regulated by Rec8p through its role in maintaining centromeric cohesion. Loss of Spo13p, and subsequent reduction in Rec8p levels at centromeres, may cause a reduction in the assembly of this complex, which may result in loss of Spo11p on chromosomes and a reduction in the formation of stable interchromosomal interactions necessary for rereplication initiation.
Finally, we have uncovered a potential role for Cdc55p-dependent PP2A during premeiotic DNA replication. Cells lacking Cdc55p express IME1
but fail to complete meiotic replication (L. Rice and J. T. Nickels, unpublished data). How might Cdc55p regulate meiosis? CDC55
was originally discovered in a screen aimed at isolating recessive mutations causing cs
morphology defects; cdc55
cells have multiple elongated buds that are defective in cytokinesis (14
). The cs
phenotype can be suppressed by expressing a dominant allele of CDC28
, lacking the conserved inhibitory tyrosine phosphorylation site (Y19) (30
). Thus, Cdc55p may regulate the Cdc28p/Clb activity required for meiotic replication through regulating the degree of inhibitory phosphorylation at Y19. It may do so indirectly through regulating the phosphorylation state and/or stability of Swe1p, as this kinase accumulates in mitotically grown cdc55
cells treated with microtubule-destabilizing agents that cause a G2
/M arrest (54
Cdc55p may regulate Cdc45p, which is a component of the pre-RC that is required for replication initiation (19
). Xenopus laevis
eggs depleted of PP2A have less Cdc45 bound to chromatin (5
). Cdc55p may also regulate meiosis through its regulation of protein degradation. The loss of cdc55
can suppress the ts
phenotype of cdc20-1
). Cdc20p is an activator of the anaphase-promoting complex (APC/C), and it interacts with and activates a phosphorylated form of the APC (55
). Moreover, cdc55
is synthetic lethal with loss of a specific F-box protein, Grr1p, known to regulate the SCF ubiquitin-ligase complex (20
). These results argue that Cdc55p may function to regulate protein proteolysis through its regulation of SCF and/or APC/C complex activities. In any event, our data support the idea that Cdc55p-dependent PP2A activity is essential during meiosis, and we are currently examining how it regulates the meiotic process.