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Resistance to DNA Damage Conferred by Pap1 Despite Inhibition of Chk1

by
Carrie P. Belfield
B.S., Delaware State University - May 2004


Thesis Advisor: Nancy Walworth, Ph.D.
Graduate Program in Cellular & Molecular Pharmacology

Pharmacology Department Conference Room
4th Floor, RWJMS Research Tower
Piscataway

Wednesday, September 5, 2012
10:00 a.m.


Abstract

Fission yeast cells utilize different checkpoint pathways throughout the cell cycle to ensure their genome integrity and survival. The protein kinase Chk1 has been found to be a key component of the DNA damage checkpoint pathway. Upon exposure to agents that result in DNA damage, Chk1 is phosphorylated and activated, followed by its accumulation in the nucleus which has been shown to be regulated through its C-terminal domain. Studies of a C-terminal specific allele of Chk1, D469G, have shown that this mutant is partially active when expressed in multiple copies, but not when expressed at endogenous levels. The goal of this thesis was to identify and characterize DNA damage specific suppressors of Chk1D469G. To do this, we designed a genetic screen utilizing a genomic library of plasmids where we identified a gene, Pap1, which was able to induce resistance to DNA damaging agents, as well as improve survival in the presence of other types of damage. Through this work, we have shown that multi-copy expression of Pap1 can suppress the growth defect of chk1-deficient cells in the presence of DNA damaging agents such as CPT and HU, or in the presence of unligated DNA or DNA replication elongation defects. However, Pap1 was not able to compensate for loss of chk1 in cells undergoing mitotic catastrophe nor those exposed to ultraviolet light. Thus, Pap1 exhibits selectivity for particular types of DNA damage. A set of Pap1 mutants helped us to determine that its localization to the nucleus, as well as transcriptional activity is required to induce survival in the presence of DNA damage. Taken together, these results lead us to believe that Pap1 may upregulate the transcription of some gene(s) that either help repair DNA damage or aid in stabilizing cells until the damage can be repaired in the next round of the cell cycle.


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