hypomutable and fail to respond normally to signals generated by stalled replication forks. Therefore, Cdc7p-Dbf4p is emerging as perhaps a more global regulator of chromosome maintenance and stability than previously thought.
increased life span in yeast and Caenorhabditis elegans. SIR2 encodes a histone-dependent deacetylase and has at least seven orthologues in human cells. No evidence has been reported that the Sir proteins influence replication globally, as our data suggest. We are testing whether the Sir proteins act directly at origins of replication and negatively regulate initiation events. If this is occurring, it could provide a mechanism for the establishment of transcriptional or developmental states that were coupled to replication of certain chromosomal domains.
For this reason we are studying this protein both in yeast and in human cells. We have generated wild-type human cDNA clones and have constructed baculoviruses for purification of both the human and yeast enzymes. We have raised monoclonal antibodies against human Cdc7 and are now raising antibodies against the Dbf4 subunit. We hope to gain valuable reagents for examining the regulation and localization of the human kinase, both during the normal cell cycle and during periods of genomic stress. The Dbf4 protein has two classical D-box motifs and also a KEN-box motif. Both of these sequences are known to promote polyubiquitylation and proteasome-dependent degradation of cyclins and other unstable proteins. We are examining if these sequences function similarly in the human and yeast kinases. We are most interested in determining the role of the Cdc7p-Dbf4p kinase during periods of DNA damage or replication-fork arrest. Both the human and yeast Dbf4 proteins contain a single BRCT domain at the amino terminus. BRCT domains (first defined as a tandem repeat at the C-terminus of BRCA1) are present in a large family of proteins involved in DNA repair. Published and unpublished work indicates that yeast Cdc7pDbf4p is an important target of the S-phase checkpoint. The S-phase checkpoint in yeast responds to stalled replication forks that occur through a variety of insults. Since abrogating checkpoints are thought to facilitate tumorigenesis, we are examining if the human Cdc7 kinase is similarly a target of checkpoint kinases following DNA damage. Also, we are taking a genetic approach in yeast to more accurately determine its effect on DNA repair and replication.
Figure 1. S. cerevisiae replication cycle
We are also studying the Cdc7p-Dbf4p kinase, which is a conserved, two-subunit serine/threonine protein kinase required for a late step in replication initiation. We are interested in understanding the regulation of Cdc7p-Dbf4p kinase activity and determining its critical in vivo substrates. Cdc7p subunit abundance is constant throughout the cell cycle, but the Dbf4p subunit is cyclically expressed and is degraded during mitosis. The Cdc7p-Dbf4p kinase is required for DNA replication, but it has less-well-defined roles in promoting error-prone DNA repair and progression through meiosis. In response to DNA damage, Dbf4p is phosphorylated in a checkpoint-dependent manner and this decreases Cdc7p-Dbf4p kinase activity. CDC7 mutants are
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