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2002 Scientific Report

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Laboratory of Chromosome

Laboratory of Chromosome Replication Michael Weinreich, Ph.D. Dr. Weinreich received his Ph.D. in biochemistry from the University of Wisconsin–Madison in 1993. He then served as a Postdoctoral Fellow in the laboratory of Bruce Stillman, director of the Cold Spring Harbor Laboratory, New York, from 1993 to 2000. Dr. Weinreich joined VARI as a Scientific Investigator in March 2000. Staff Andrei Blokhin, Ph.D. Don Pappas, Ph.D. Carrie Gabrielse, B.S. Marleah Russo, B.S. Laboratory Members Student Ashley Mynsberge Research Projects O ne critical step after the commitment to cell division is chromosome replication. Our laboratory is interested in understanding how the initiation of DNA replication occurs at the molecular level and how initiation events at each chromosomal origin are restricted to once per cell cycle. As cells exit mitosis and enter the G1 phase, they assemble a “pre-replicative complex” (pre-RC) at multiple replication origins. Additional lesswell-defined complexes formed at the origin in G1 are then activated to form bidirectional replication forks within a very short time (S-phase). Restricting the assembly of pre-RCs to G1 is a key regulatory event insuring that replication origins become competent only after completion of the previous cell cycle. In Saccharomyces cerevisiae, cyclin-dependent kinases inhibit pre-RC formation throughout the cell cycle, but as their levels fall during exit from mitosis, pre-RCs are able to form. Eukaryotic origins of replication have been precisely defined only in budding yeast. An initiator protein (ORC) has also been extensively characterized. ORC is a six-subunit complex that recognizes conserved sequence elements in all origins and is required for the initiation of replication. ORC is bound to origins throughout the cell cycle; however, in late mitosis, Cdc6p binds to ORC and promotes loading of the MCM complex (a helicase) at the origin. ORC, Cdc6p, and the MCM complex are required to form the pre-RC in vivo. Subsequent events occurring in G1 are much less understood, including the association of Cdc45p, the loading of DNA polymerases, and the activation of replication by cyclin-dependent kinases and the Cdc7 protein kinase. Our long-term goal is to define the protein components of the replication complexes that form in G1, and particularly to understand how complex assembly is regulated. Cdc6p is a critical limiting factor for assembly of the pre-RC. We have previously shown that Cdc6p interacts with ORC and that its essential activity requires a functional ATP-binding domain. Cdc6p also couples replication initiation with progression through the remainder of the cell cycle. If initiation does not occur, a nonessential N-terminal domain of approximately 50 amino acids is required for preventing a “reductional mitosis,” in which the unreplicated chromosomes are randomly segregated. Cdc6p very likely regulates passage through mitosis by inhibiting cyclindependent kinases. We have taken a genetic approach to identifying additional factors that are important for initiation. Using a temperature-sensitive mutation in CDC6, we have isolated a number of dosagedependent and extragenic suppressors that restore growth at high temperature. These suppressors define several novel pathways influencing replication. For example, we isolated one class of extragenic suppressors that contained mutations in the silent information regulators SIR2, SIR3, and SIR4. The Sir proteins are required for the formation of heterochromatic regions at the silent mating-type loci and at telomeres. In addition, Sir2p suppresses recombination at the rDNA locus and promotes 53

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. 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 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. 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 Cdc7p- Dbf4p 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. 54

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