Academic journal article Genetics

Novel Role for Checkpoint Rad53 Protein Kinase in the Initiation of Chromosomal DNA Replication in Saccharomyces Cerevisiae

Academic journal article Genetics

Novel Role for Checkpoint Rad53 Protein Kinase in the Initiation of Chromosomal DNA Replication in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

A novel role for Rad53 in the initiation of DNA replication that is independent of checkpoint or deoxynucleotide regulation is proposed. Rad53 kinase is part of a signal transduction pathway involved in the DNA damage and replication checkpoints, while Cdc7-Dbf4 kinase (DDK) is important for the initiation of DNA replication. In addition to the known cdc7-rad53 synthetic lethality, rad53 mutations suppress mcm5-bob1, a mutation in the replicative MCM helicase that bypasses DDK's essential role. Rad53 kinase activity but neither checkpoint FHA domain is required. Conversely, Rad53 kinase can be activated without DDK. Rad53's role in replication is independent of both DNA and mitotic checkpoints because mutations in other checkpoint genes that act upstream or downstream of RAD53 or in the mitotic checkpoint do not exhibit these phenotypes. Because Rad53 binds an origin of replication mainly through its kinase domain and rad53 null mutants display a minichromosome loss phenotype, Rad53 is important in the initiation of DNA replication, as are DDK and Mcm2-7 proteins. This unique requirement for Rad53 can be suppressed by the deletion of the major histone H3/H4 gene pair, indicating that Rad53 may be regulating initiation by controlling histone protein levels and/or by affecting origin chromatin structure.

ELABORATE regulatory mechanisms have evolved in eukaryotic cells to ensure that DNA replication occurs only once per cell cycle (for recent reviews see Bell and Dutta 2002; Diffley 2004). First, a multiprotein prereplication complex (pre-RC) is assembled onto origins of replication during the G1 phase of the cell cycle. Origins are bound throughout the cell cycle by a six-member protein complex known as the origin recognition complex (ORC). Cdc6, which is produced in the G1 phase, together with Cdt1 protein loads the multimeric minichromosomemaintenance (MCM) complex onto the ORC. The pre-RC is then activated by two independent protein kinases: cyclin-dependent kinase (CDK) (Cdk1-Clb) and Dbf4-dependent kinase (DDK) (Cdc7-Dbf4). Both CDK and DDK are activated in late G1 phase by the binding of unstable regulatory subunits, the Clb5,6 and Dbf4 proteins, respectively.

Both CDK and DDK are needed for Cdc45 protein to load DNA polymerases and other replication proteins onto origins during S-phase (ZOU and STILLMAN 1998; APARICIO et al. 1999). DDK phosphorylates Mcm2 in vitro (and likely in vivo) as part of the MCM complex (LEI et al. 1997; OSHIRO et al. 1999; WEINREICH and STILLMAN 1999). Because the MCM complex is believed to act as the replicative helicase (LABIB and DIFFLEY 2001), it is thought that inhibition of the helicase activity is abolished by phosphorylation, perhaps by allosteric change of the MCM complex (SCLAFANI et al. 2002, 2004; FLETCHER et al. 2003; CHEN et al. 2005). Hence, both events are necessary for producing the binding of DNA replication proteins and fork movement (JARES et al. 2000; LEI and TYE 2001). The essential DDK step in DNA replication initiation can be bypassed in budding yeast cells carrying the mcm5-bob1 mutation (JACKSON et al. 1993; HARDY et al. 1997), which produces constitutive Cdc45 loading (SCLAFANI et al. 2002). In this regard, the mcm5-bob1 encoded protein may mimic the allosteric change required for the MCM helicase to become an active complex (SCLAFANI et al. 2002, 2004; FLETCHER et al. 2003; CHEN et al. 2005). At the same time, additional CDK activity is still needed to initiate DNA replication. Thus, cells that carry either a clb2D or a clb5D mutation are ineffective in mcm5-bob1-dependent bypass of DDK (SCLAFANI et al. 2002). In this manner, clb2D and clb5D mutations suppress the mcm5-bob1 mutation (SCLAFANI et al. 2002).

Concomitantly, DNA checkpoint mechanisms have evolved to monitor the successful completion of cell cycle events involving DNA replication and mitosis (see reviews in FOIANI et al. 2000; NYBERG et al. 2002; KASTAN and BARTEK 2004). …

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