Research Description:
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When eukaryotic cells are exposed to agents that cause DNA damage,
such as UV light, X-rays or drugs used in cancer chemotherapy, progression
through the cell cycle is transiently arrested. Research in
our laboratory is directed toward understanding the mechanism through
which eukaryotic cells arrest the cell cycle in response to DNA
damage. We use the fission yeast, Schizosaccharomyces pombe,
as a model system for these studies since the molecules which regulate
cell cycle progression have been well-characterized in this organism,
and since yeast is amenable to both classical and molecular genetic
analysis. Furthermore, molecules that regulate the cell cycle
are well-conserved throughout evolution such that many enzymes encoded
by human genes can function in yeast. The signal
transduction pathway that couples the detection of DNA damage
to control of cell cycle progression has been described as the
DNA damage checkpoint. We have focused on the role played
by a conserved protein kinase encoded by the chk1 gene of fission
yeast in this signal transduction process. Cells which lack
chk1 function are unable to arrest the cell cycle when DNA damage
takes place, enter mitosis with damaged DNA and subsequently die.
To characterize the role of p56chk1 in mediating cell cycle arrest
following DNA damage, two complementary lines of research are
being carried out: biochemical studies to characterize the activity
of p56chk1 and its response to DNA damage; and genetic screens
to identify additional proteins which act with p56chk1 to mediate
cell cycle arrest. Recently, it has been demonstrated that
patients with defects in a gene related to a yeast cell cycle
checkpoint gene are at increased risk for cancer. Dissection
of the chk1-dependent DNA damage checkpoint in fission yeast will
foster our understanding of how eukaryotic cells respond to DNA
damage and how defects in this response may contribute to the
development of cancer.
We have established that the p56chk1 protein is phosphorylated
in response to DNA damaging agents. This phosphorylation
is dependent on the activity of several other gene products that
are required for cell cycle arrest in response to DNA damage.
Phosphorylation of p56chk1 correlates with cell cycle arrest and
dephosphorylation accompanies recovery of cell cycle progression
following DNA repair. We have identified several proteins
that physically interact with p56chk1 and are in the process of
characterizing them. The association of two of these proteins,
Rad24 and Rad25, is stimulated dramatically in response to DNA
damage. We have identified the domain of p56chk1 that is
important for this interaction and mutations within that domain
affect p56chk1 function. We have developed a genetic screen
using the anti-cancer drug camptothecin for the identification
of novel checkpoint defective alleles of the chk1 gene.
These mutants are being used in genetic screens designed to identify
additional components of the DNA damage checkpoint pathway.
Recent Publications
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Walworth, N., S. Davey and D. Beach. 1993. Fission yeast chk1
protein kinase links the rad checkpoint pathway to cdc2. Nature
363:368-371.
Den Haese, G. J., N. Walworth, A. M. Carr and K. L. Gould. 1995.
The wee1 protein kinase regulates T14 phosphorylation of fission
yeast cdc2. Molecular Biology of the Cell 6:371-385.
Walworth, N. C. and R. Bernards. 1996. rad-Dependent response
of the chk1-encoded protein kinase at the DNA damage checkpoint.
Science 271:353-356.
Lindsay, H. D., D. J. F. Griffiths, R. J. Edwards, P. U. Christensen,
J. M. Murray, F. Osman, N. Walworth and A. M. Carr. 1998. S-phase
specific activation of Cds1 kinase defines a subpathway of the
checkpoint response in S. pombe. Genes and Development 12:382-395.
Walworth, N.C. 1998. Rad9 comes of age. Science. 281:185-186.
Chen, L., Liu, T-H., and N. C. Walworth. 1999. Association of
Chk1 with 14-3-3 proteins is stimulated by DNA damage. Genes and
Development 13:675-685.
Wan, S., H. Capasso and N. C. Walworth. 1999. The topoisomerase
I poison camptothecin generates a Chk1-dependent DNA damage checkpoint
signal in fission yeast. Yeast 15:821-828.
O’Connell, M.J., N. C. Walworth, and A. M. Carr. 2000. The G2-phase
DNA-damage checkpoint. Trends in Cell
Biology. 10:296-303.
Walworth, N.C. 2000. Cell-cycle checkpoint kinases: checking
in on the cell cycle. Current Opinion in Cell Biology.
12:697-704.
Wan, S. and N. C. Walworth. 2001. A novel genetic screen yields
checkpoint defective alleles of
Schizosaccharomyces pombe chk1. Current Genetics 38:299-306.
Walworth, N.C. 2001. DNA damage: Chk1 and Cdc25, more than meets
the eye. Current Opinion in Genetics and
Development 11:78-82.
Liu, H.Y., B. S. Nefsky and N. C. Walworth. 2002. The Ded1 DEAD box helicase
interacts with Chk1 and Cdc2. J. Biological Chemistry, 277:2637-2643.
Capasso, H., C. Palermo, S. Wan, H. Rao, U. P. John, M. J. O’Connell, N. C. Walworth. 2002.
Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest. J.
Cell Science,115:4555-4564.
Colon-Berlingeri, M. and N. C. Walworth. 2003. Use of in vivo Gap Repair for Isolation of
Mutant Alleles of a Checkpoint Gene, in Methods in Molecular Biology, vol. 241: Cell Cycle
Checkpoint Control Protocols. pages 175-187. H. B. Lieberman, Ed. Humana press Inc., Totowa, NJ.
Ahmed, S., C. Palermo, S. Wan, and N. C. Walworth. 2004. A novel protein with similarities
to Rb binding protein 2 compensates for lack of Chk1 function and affects histone modification
in fission yeast. Molecular and Cellular Biology 24(9):3660-3669.
Dunaway, S. and N. C. Walworth. 2004. Assaying the DNA Damage Checkpoint in Fission Yeast.
Methods. 33:260-263.
Palermo, C. and N. C. Walworth, in press. Yeast as a model for studying cell cycle
checkpoints, in Yeast as a Tool in Cancer Research. J. Heitman and J. L. Nitiss, ed.
Kluwer Academic Publishers.
Palermo, C. and N. C. Walworth, in press. Assaying Cell Cycle Checkpoints: Activity
of the Protein Kinase Chk1, in Methods in Molecular Biology, T. Humphrey, ed. Humana Press Inc.
Lab Staff
| Shakil Ahmad |
Research Teaching Specialist |
| Barbara Dul |
Graduate Student |
| Stephanie Vnadi |
Research Teaching Specialist V |
| Carmela Palermo |
Graduate Student |
| Hui Rao |
Research Teaching Specialist |
|
