ATM Activation by Oxidative Stress

TLDR: It is shown that oxidation of ATM directly induces ATM activation in the absence of DNA DSBs and the MRN complex, and that ATM is an important sensor of reactive oxygen species in human cells.

Abstract: The ataxia-telangiectasia mutated (ATM) protein kinase is activated by DNA double-strand breaks (DSBs) through the Mre11-Rad50-Nbs1 (MRN) DNA repair complex and orchestrates signaling cascades that initiate the DNA damage response. Cells lacking ATM are also hypersensitive to insults other than DSBs, particularly oxidative stress. We show that oxidation of ATM directly induces ATM activation in the absence of DNA DSBs and the MRN complex. The oxidized form of ATM is a disulfide-cross-linked dimer, and mutation of a critical cysteine residue involved in disulfide bond formation specifically blocked activation through the oxidation pathway. Identification of this pathway explains observations of ATM activation under conditions of oxidative stress and shows that ATM is an important sensor of reactive oxygen species in human cells.

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Copyright
by
Zhi Guo
2008

The Dissertation Committee for Zhi Guo certifies that this is
the approved version of the following dissertation
ATM Activation by Oxidative Stress
Committee:
Tanya Paull, Supervisor
Kelvin Dalby
George Georgiou
Jon Huibregtse
David Johnson

ATM Activation by Oxidative Stress
by
Zhi Guo, B.S.; M.S.
Dissertation
Presented to the Faculty of the Graduate School of
The University of Texas at Austin
in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
The University of Texas at Austin
August 2009

iv
Acknowledgements
I would like to express my gratitude to Dr.Tanya Paull for her constant guidance
and motivation towards the successful completion of my degree. Sincere thanks to my
committee members: Drs. Kelvin Dalby, George Georgiou, Jon Huibregtse and David
Johnson for providing me with insightful comments. I would like to express my heartfelt
thanks to all my family and friends without whom this study would have been extremely
difficult and incomplete.

v
ATM Activation by Oxidative Stress
Zhi Guo, Ph.D.
The University of Texas at Austin, 2009
Supervisor: Tanya T. Paull
The Ataxia-telangiectasia mutaed (ATM) protein is regarded as the major regulator
of the cellular response to DNA double Strand Breaks (DSBs). In response to DSBs,
ATM dimers dissociates into active monomers in a process promoted by Mre11-Rad50-
Nbs1 (MRN) complex. ATM-deficient cells exhibit signs of chronic oxidative stress,
suggesting that ATM plays an important role in the regulation of reactive oxygen species
(ROS). I show for the first time that ATM can be activated by oxidative stress directly in
the form of exposure to H
2
O
2
. In vitro kinase assays with purified ATM suggest that the
activation by H
2
O
2
is independent of DSBs and the MRN complex. In 293T cells, H
2
O
2
induces ATM autophosphorylation on serine 1981. p53 and Chk2 are also phosphorylated
by ATM after H
2
O
2
treatment but not histone H2AX and heterochromatin protein Kap1,
indicating that ATM activation by H
2
O
2
in human cells is independent of DNA damage. I
also show that the cysteine residue 2991 is critical for ATM activation by H
2
O
2
in vitro.