Faculty

Research Interest

Control of DNA replication Initiation in Fission Yeast

Long-term goals of our laboratory are to understand the molecular mechanisms of DNA replication initiation and to determine how defects in initiation are coupled to a checkpoint control that prevents mitosis in the absence of a complete round of DNA replication. Initially, we cloned the gene corresponding to the cell cycle mutant cdc20+ in fission yeast and demonstrated that it encoded the large catalytic subunit of DNA polymerase epsilon (Pol epsilon; D'Urso and Nurse, 1997). This gene when mutated results in a cell cycle arrest in late G1 or early S phase, suggesting that DNA replication is impaired. Our further analysis of Pol epsilon and its associated subunits suggests that Pol epsilon plays a critical role in the assembly of the replication initiation complex (Feng et al., 2003). The N-terminal catalytic domains of Pol epsilon are dispensable for cell viability. We have also discovered that the polymerase domains associated with the catalytic subunit of Pol epsilon are dispensable for cell viability (Feng and D'Urso, 2001). Although cells are viable in the absence of the polymerase domains they experience a pronounced cell cycle delay. We discovered that this delay, presumably imposed by a replication defect, reflects activation of a novel checkpoint control that blocks mitosis. Unlike other DNA replication-associated DNA damage checkpoints, which activate the Cds1 kinase, cells lacking the polymerase domains of Pol epsilon activate the checkpoint kinase, Chk1. We have also shown that this same checkpoint is activated in mutants defective in Orc1, Cdc18, and Orc5, leading us to propose that mutants defective in DNA replication initiation (called rid mutants) activate this potentially novel checkpoint.

Cloning of Dpb2, Dpb3 and Dpb4

We have also cloned the three additional subunits of Pol epsilon and have shown that two of these are essential for cell viability. Mutations in Dpb2 result in an early S phase arrest consistent with this protein having a role in the early events of DNA replication initiation. On the other hand, loss of Dpb3 does not result in any obvious defects in DNA replication, but cells still arrest in either late S phase or G2. They also display a peculiar phenotype in that many cells arrest with two or more nuclei, suggesting that Dpb3 might play some role in later stages of cytokinesis.

Apoptosis in Yeast

In related studies, we have recently shown that cells defective in DNA replication initiation can undergo a primitive form of apoptosis or programmed cell death (PCD) (Burhans et al., 2003). In addition to the work on DNA replication, we also found that defects in RNA splicing might also result in cell death that is partially suppressed by mutations in the Wee1 kinase (Oltra et al., 2004). We are currently testing the hypothesis that active cell death can occur in a single-celled eukaryotic organism like yeast in response to a variety of cell cycle blocks. Our goal is to determine whether PCD occurs in fission yeast, and if it does, to genetically dissect the pathways that influence activation of the PCD program.

Funding Provided by:

American Cancer Society
American Heart Association
National Cancer Institute, NIH

Lab Members

Andrew Phillips, Ph.D., Postdoctoral Fellow
Julie Sierra-Montes, Ph.D., Postdoctoral Fellow
Alexandra Locovei, Graduate Student
Ling Yin, Graduate Student

Selected Recent Publications

Spiga, G. and D'Urso, G. (2004) Identification and cloning of two putative subunits of DNA polymerase epsilon in fission yeast. Nucleic Acids Research, 32, 4945-4953.

Rodriguez-Menocal, L. and D'Urso, G. (2004) Programmed cell death in fission yeast. FEMS Yeast Research, 5, 111-117.

Oltra, E., Verde, F.,Werner, R. and D'Urso, G. (2004). A novel RING-finger-like protein Ini1 is essential for cell cycle progression in fission yeast. J. Cell Science, 117, 967-974. 

Burhans, W. C., Weinberger, M., Marchetti, M. A., Ramachandran, L., D'Urso, G. and Huberman, J. A. (2003). Apoptosis-like yeast cell death in response to DNA damage and replication defects. Mutat Res 532, 227-43.

Feng, W., Rodriguez-Menocal, L., Tolun, G. and D'Urso, G. (2003). Schizosacchromyces pombe Dpb2 binds to origin DNA early in S phase and is required for chromosomal DNA replication. Mol Biol Cell 14, 3427-36.

Feng, W. and D'Urso, G. (2001). Schizosaccharomyces pombe cells lacking the amino-terminal catalytic domains of DNA polymerase epsilon are viable but require the DNA damage checkpoint control. Mol Cell Biol 21, 4495-504.

D'Urso, G. and Nurse, P. (1997). S. pombe cdc20+ encodes DNA polymerase ε is required for the chromosomal replication but not for the S phase checkpoint. Proc. Natl. Acad. Sci. USA 94, 12491-12496.