3. RESULTADOS Y DISCUSIÓN
3.5 PRECIO DE LA LECHE
Two distinct checkpoints that regulate mitosis in response to the DNA status of the cell exist in both budding and fission yeast; one that monitors the completion of DNA replication and one that monitors DNA damage. In order to be effective, a checkpoint needs to be able to monitor changes to nuclear material, to transmit this information to the cell cycle machinery and to have a target which will take appropriate action once this signal is passed on.
1.10.1 Checkpoint gene functions
1.10.2
radlhus
genesSix genes have been identiHed that have an essential role in the operation of the DNA replication and DNA damage checkpoints: radl, radS, rad9, radl 7, rad26 and husl (Sheldrick and Carr, 1993). All six checkpoint genes are non-essential for mitotic growth under normal conditions. They were identified on the basis of their radiation or hydroxyurea sensitivity. Genetic analysis has shown that mutations at all six loci were unable to cause cell cycle arrest following treatment with either DNA damaging agents or hydroxyurea, radlhus gene products may form a structure that interacts with replication complexes, generating a signal to inhibit initiation of mitosis. Although the molecular mechanisms of this pathway
remain to be elucidated, it is thought to operate via the tyrosine phosphorylation of cdc2 (Sheldrick and Carr, 1993).
1.10.3
chkl
geneThere appears to be a separate checkpoint pathway to prevent cells which have passed Start, but not initiated DNA replication, from entering mitosis. This involves the chkl gene which encodes a putative protein kinase that arrests cell cycle progression when overexpressed (Ford et a/., 1994). Chkl was isolated as a suppressor of a cold-sensitive allele of cdc2 and it has therefore been implicated in the regulation of p34odc2 kinase activity (Walworth et al., 1993). Experiments in which the mammalian CDI, p 2 1 c iP i was expressed in S. pombe pointed to a
role for PCNA in the chkl pathway (Toumier et a l, 1996). In the presence of DNA damage, p56Chki becomes phosphorylated, this modification being
abolished in the catalytic domain, suggesting that an autophosphorylation process (Walworth and Bernards, 1996). Carr et al. (1995) have demonstrated a second checkpoint role for ch kl, namely to prevent entry into mitosis in cells compromised for passage through Start. This pathway also requires radl 7, so it is related to, but distinct from, the DNA damage checkpoint (Carr et a l, 1995).
1.10.4
cdcl8, cut5lrad4 and cdtl
genesThe mechanism by which the cell can monitor completion of S phase is thought to operate through checks on DNA replication. This hypothesis is supported by observations from fission yeast that cdcl8 (Kelly et a l, 1993), cdtl (Hoffman and Beach, 1994) and cut5lrad4 (Saka and Yanagida, 1993) are required both to initiate S phase and to block M phase until DNA replication is complete. Cells lacking these genes will block in G l for a short interval before proceeding into S
phase. There may be a temporal ordering of checkpoint controls which monitors the progression of a cell through the cell cycle as it has been demonstrated that the p34odc2-cyclin B complex is important in maintaining the dependency of M phase on S phase (Hayles et a/., 1994).
cdc 18 is required for DNA replication and was originally identified as a gene that was necessary for completion of the cell cycle (Nasymth and Nurse, 1981). It was later cloned by direct genetic complementation and as a multicopy suppressor of the cdclO gene (Kelly et a i, 1993). cdcIS exerts its function at the Start control through transcriptional activation of the cdcI0lresl/res2 complex. A biochemical role for cdclS has not yet been identified, although a nucleotide-binding site is present suggesting potential ATPase activity. The gene shows 28% similarity in amino acid sequence to CDC6 in S. cerevisiae^ a gene required for onset of S phase which may also interact with origins of replication (Hogan and Koshland, 1992; Li and Herskowitz, 1993; Liang et aL, 1995). In addition, Xenopus and human proteins closely related to Cdcl8/Cdc6 have recently been identified (Williams gf a/., 1996).
Transcripts levels of cdcIS vary in the cell cycle and peak at the G l/S transition (Kelly et a/., 1993). Ectopic expression of cdcl8 will rescue temperature sensitive cdcIO mutants but not cells deleted for cdclO, indicating that cd c l8 is an important link between the G l transcriptional control and the initiation of DNA replication. It has been proposed that cdcI8 is actively involved in the initiation of DNA replication at the onset of S phase and evidence for this role was demonstrated when overexpression of cd c l8 induced DNA synthesis in the absence of mitosis (Nishitani and Nurse, 1995). Recently, evidence was presented that the mitotic-Cdk complex affects DNA replication by inhibiting the accumulation and thereby the function of cd cl8 (Jallepalli and Kelly, 1996). It is
thought that cdc 18 may have an additional role in the checkpoint control at M phase (Kelly et a i, 1993).
The
cuts
gene was isolated as a conditional mutant that enters mitosis having failed to complete DNA synthesis, suggesting that the checkpoint linking completion of DNA synthesis to mitosis was defective (Saka and Yanagida, 1993).cuts
is an allele of rad4, a gene involved in DNA repair and has homology to human RCCl.The cdtl gene was cloned ^ a potential target of the DSC-1 transcription factor (Hoffman and Beach, 1994). Like cdc 18 and cutSy cdtl is also required for DNA synthesis acting as a checkpoint coupling completion of DNA replication to the onset of mitosis. Transcription of cdtl is variable through the cell cycle, peaking at the G l/S transition point (Hoffman and Beach, 1994). To date, there are no known homologues of cdtl in other organisms.
1.10.5 Preventing entry into mitosis from G l
It is important that mitotic initiation is prevented whilst the cell is in the pre-Start phase of the cell cycle. The ruml gene is thought to have a key role in this process. Although ruml is also involved in preventing mitosis during G 1, it is not thought that the ruml and chkl genes operate through the same pathway (Moreno and Nurse, 1994; Carr et a i, 1995). The mitotic arrest caused by chkl overexpression is not dependent on ruml gene function (Ford et a/., 1994) and vice versa y {Czn et a/., 1995), an indication that ruml is not activated by a chkl- dependent pathway following DNA damage (Carr et a i, 1995).
A fission yeast mutation in a gene related to the mammalian gene RCCl (repressor of chromosome condensation) is also known to affect entry into mitosis (Matsumoto and Beach, 1991). The mutation itself lies in the pim l/d cd l gene which encodes a protein with 30% sequence identity to RCCl. Mutant cells can enter mitosis without passing Start and will accumulate in M phase at the non- permissive temperature. Removal of p im l activity from S. pombe cells results in an inability to décondense chromosomes after mitosis (Sazer and Nurse, 1993). Unlike the situation in mammalian cells, p im l activity is not required for DNA replication and loss of activity does not result in the dependency of mitosis on completion of DNA synthesis to be abolished (Dasso et al., 1992). Recently it was shown that following mitosis, fragmentation of the nuclear envelope occurs in p im l/d c d l mutants (Demeter et a l, 1995). This suggests pim l may be necessary for the re-establishment of nuclear structure following mitosis in S. pombe.