DNA REPLICATION CHECKPOINTS IN FISSION YEAST

Gennaro D’Urso,
University of Miami School of Medicine, Department of Biochemistry and Molecular Biology, 1011 NW 15th St., Miami, Florida. gdurso@miami.edu

Initiation of DNA replication requires the ordered assembly of macromolecular complexes at replication origins (Diffley and Labib, 2002).  The first step in the assembly of the initiation complex involves the binding of MCM proteins to ORC-associated chromatin during the G1 phase of the cell cycle to form pre-Replicative Complexs (pre-RCs).  Binding of DNA polymerases and other accessory proteins to pre-RCs follows this step and is required to support both primer synthesis and strand elongation.  Activation of DNA synthesis at the G1 to S phase transition requires both Cdk and Cdc7 kinase activities.  These kinases are believed to induce a conformational change within the pre-RC that allows loading of DNA polymerases and final assembly of the replication complex (Sclafani et al., 2002).

Assembly of a functional replication fork is crucial for the transition from G1 into S phase, and is believed to play a pivotal role in checkpoint signaling (D'Urso et al., 1995b; Tercero et al., 2003).  Mutations that disrupt this complex have profound effects on cell cycle progression, and in some cases, cause cells to enter mitosis in the absence of DNA replication.  Although components of the replication fork might be directly involved in checkpoint signaling in S phase, there is currently no direct evidence to support this hypothesis.  We showed previously that fission yeast cells deleted for the gene encoding DNA polymerase alpha (Pol a) initiate mitosis in the absence of DNA replication indicating that Pol a might be important for checkpoint signaling (D'Urso et al., 1995a).   Moreover, deletion of the gene encoding Cdc18, a protein required for assembly of the pre-Replicative Complex results in a similar phenotype (Kelly et al., 1993).  One explanation for these results is that in the absence of a functional replication complex, cells are incapable of sending the checkpoint signal that normally responds to either DNA damage or stalled forks.  However, it is still possible that a checkpoint exists that monitors assembly of replicative complexes before they are converted into active replication forks.  This signal might be dependent on the loading of Pol a or other replication proteins to chromatin in late G1 or early S phase.  Interestingly, in budding yeast, some cdc7 mutants arrest prior to DNA replication initiation, and this arrest occurs independently of any known checkpoint pathway (Weinert and Hartwell, 1993; Weinert et al., 1994).  This implies a novel checkpoint may function to block entry into mitosis from late G1, even in the absence of a functional replication fork(Toyn et al., 1995). 

We have used fission yeast as a model system to identify and characterize genes that are required for DNA replication initiation.  We initially cloned the gene corresponding to the cell cycle mutant cdc20 which arrests in lateG1/early S phase upon shift to the restrictive temperature (D'Urso and Nurse, 1997).  We demonstrated that cdc20 encodes the large catalytic subunit of DNA polymerase epsilon (Pol e), suggesting that Pol e is required for an early step in DNA replication.  Further analysis of Pol e and its associated subunits in our laboratory has confirmed that Pol e is essential for DNA replication initiation (Feng and D'Urso, 2001; Feng et al., 2003).  Our observation that the polymerase domains of Pol e are dispensable for cell viability, while the C-terminal half of the protein (with no known biochemical function) is absolutely essential, suggests that Pol e is required during assembly of the initiation complex.  Consistent with this idea, we have shown that both Pol e its second largest subunit, Dpb2, bind specifically to replication origins at the beginning of S phase (Feng et al., 2003).  

Analysis of mutants deleted for the N-terminal half of Pol e (cdc20∆Nterm) has revealed the existence of a potentially novel checkpoint control that is activated in response to defects in DNA replication initiation.  Mutants lacking the N-terminal half of Pol e have a cell cycle delay consistent with the activation of a checkpoint control.  Interestingly we found that this checkpoint is essential for cell viability, and is dependent on Chk1- rather than Cds1-kinase.  This result was surprising because most replication mutants grown under semi-permissive conditions are dependent on expression of Cds1, which is required to both stabilize replication forks (Lopes et al., 2001) and prevent premature entry into mitosis by inhibiting the mitotic activator Cdc25 (Boddy et al., 1998).  Our results imply that under conditions where Pol e activity is compromised, cells respond by activating a Chk1-dependent pathway that inhibits entry into mitosis.  We confirmed this hypothesis by complementing the cdc20∆Nterm ∆chk1 strain by integrating a temperature-sensitive version of Cdc20∆Nterm.  This strain was temperature-sensitive for growth and arrests with a characteristic mitotic catastrophe or cut phenotype at the restrictive temperature, suggesting that Chk1 is responsible for restraining mitosis in these cells (Feng and D'Urso, 2001). 

Following our observation that cdc20∆Nterm mutants activate a Chk1-dependent pathway that is required to maintain cell viability, we set out to test whether additional DNA replication mutants can delay mitosis in the absence of Cds1 or Chk1 when grown under semi-permissive conditions.  We observed that mutants defective in DNA replication initiation, including cdc18, cdc20, cdc30 and orc5 were all non-viable and checkpoint defective in the absence of Chk1 but not Cds1 when grown under conditions where any of the corresponding single mutants are viable.  In contrast, mutants defective in replication elongation, including pcn1, cdc6 and cdc17 show a dramatic loss of viability in the absence of Cds1.  These results suggest that different checkpoint pathways are activated depending on whether initiation or elongation of DNA replication is blocked.  Interestingly, in contrast to mutants defective in DNA replication elongation, mutants defective in DNA replication initiation do not show an increase in genome instability as measured by the frequency of gross chromosomal rearrangements at the ura4+ locus (Liu et al., 1999).

We propose three models to explain our results.  First mutations that disrupt DNA replication initiation result in DNA damage or other defects that are not capable of eliciting a Cds1-dependent checkpoint response in S phase.  However, this damage is detectable in G2, and readily activates the Chk1-dependent pathway.  This model implies that the signal generated in the initiation mutants is unique and Cds1-insensitive, or alternatively, is not sufficiently robust to activate the DNA damage threshold in S phase.  Our second model suggests that the repair of DNA damage that normally accumulates in S phase in response to replication errors is largely supported by the catalytic activity of Pol e.  In the absence of Pol e-associated polymerase activity, DNA repair is inefficient and ultimately leads to activation of the Chk1 response in G2.  Although there is considerable evidence that Pol e contributes to DNA repair in both yeast and human cells, this model fails to explain the checkpoint phenotype of the other initiation mutants, including cdc18 and orc5 that are not thought to have a role in DNA repair.  Finally we have considered a third model where defects in DNA replication initiation lead to a lower number of active replication origins.  In this model late stage replication initiation complexes that remain inactive might provide a checkpoint signal that delays entry into mitosis.  This delay would be dependent on expression of the Chk1 kinase.

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