Construction of His-tagged ATR Fusion Proteins: Working toward the Identification of ATR-interacting Proteins
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Authors
Sassack, Mike
Issue Date
2004
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
The protein kinase A TR is responsible for initiating signal transmission at cell
cycle checkpoints. A TR is a 303 kDa protein containing a carboxy-terminus kinase
domain related to PI-3 kinase, and is homologous to the ATM gene product in human
cells and the rad3lMECl proteins in yeast. These proteins, together with DNA-PK, are
part of the family of PI-3 kinase-related kinases (PIKKs). All members of this family
play important roles in cell cycle checkpoints and operate to permit cell survival
following many forms of DNA damage. Upon damage of DNA in eukaryotic cells, ATR
undergoes a dramatic intranuclear relocalization, translocating to nuclear foci that
represent sites of DNA damage and repair. The ultimate goals of this research are to
identify new checkpoint pathway substrates that A TR is able to phosphorylate post-DNA
damage, and to initiate the identification of potential inhibitors of A TR function. This
study commenced by dividing A TR into seven fragments. These fragments were
subsequently cloned into a vector system and transformed in order to generate A TR
fusion proteins. The induction of these fusion constructs, mediated by their screening
and conditioning, is subject to a wide variety of specifications. Because the optimal
induction conditions have yet to be discovered, fine-tuning of the experimental
techniques must continue in order to give this research a higher degree of significance.
However, once successfully induced, a series of co-transfection experiments may be
initiated, in which the activity of A TR, plus several additional proteins can be monitored
under condition of DNA damage.
Description
v, 25 p.
Citation
Publisher
Kalamazoo College
License
U.S. copyright laws protect this material. Commercial use or distribution of this material is not permitted without prior written permission of the copyright holder.