The Effect of ATR and ATM on
UVB-Induced Stem and
Progenitor Cell Death in
Arabidopsis Root Tips
Colin Tominey
Dr. John Hays
Oregon State University
25 September 2009
The DNA Damage Response
(DDR)
• DDR is an important signal transduction
pathway:
– Coordinates cell cycle transitions
– Identifies and repairs DNA damage
– Can ultimately trigger apoptosis/programmed
cell death (PCD)
• ATR and ATM are major regulators of the
DDR pathway
ATR and ATM
• ATR and ATM are kinases
• Each is activated by a particular type
of aberrant DNA structure:
– ATR responds to single-stranded DNA
– ATM responds to double-strand breaks
Apoptosis
K.A. Cimprich and D. Cortez. ATR: An Essential Regulator of Genome Integrity. Nat Rev Mol Cell Biol 9 (2008) 616-627.
DDR at Blocked Replication Forks
• Blocked replication forks are an excellent
context in which to study DDR
• Replication complexes are blocked by a
number of aberrant DNA structures:
– Interstrand crosslinks
– Chemically-adducted DNA
– Cyclobutane pyrimidine dimers (CPDs) and
other UV photoproducts
Protein Interactions
at Blocked
Replication Forks
ATR is Not Activated
ATR is Activated
ATM is Activated
R.D. Paulsen and K.A. Cimprich. The ATR pathway: Fine-tuning the fork. DNA Repair 6 (2007) 953-966.
Protein Interactions
at Blocked
Replication Forks
TLS- Trans-Lesion Synthesis (Error-Prone Bypass)
EFB- Error-Free Bypass
Hypotheses
• ATR is essential in order to utilize ErrorFree or Error-Prone Bypass.
• Both ATR and ATM are independently
capable of signaling for programmed cell
death (PCD) via SOG1
Predictions
• ATR-deficient Arabidopsis plants will
experience an increase in UVB-induced
PCD relative to wt plants due to the loss
of EFB and EPB options.
• Arabidopsis plants deficient in both ATR
and ATM will experience less UVBinduced PCD relative to wt plants due to
the loss of the SOG1-mediated apoptotic
pathway.
Experimental Design
• Irradiated 4-day-old Arabidopsis
plants (mutants and wt) with UVB
and incubated 24h in the dark
• Stained roots with propidium iodide
and counted dead stem and
progenitor cells
Stem and Progenitor Cells in Arabidopsis
Stem and Progenitor Cell Types:
Vascular
Epidermal / Lateral Root Cap
Cortical / Endodermal
Columellar / Central Root Cap
Results
5
4
3
0.3 kJ/m^2 UVB
No UVB
2
1
at
m
at
r-
wt
at
m
-
0
at
r-
Avg. Dead Stem and Progenitor Cells
per Root
UVB-Induced Stem and Progenitor Cell Death in
Arabidopsis Roots
Plant Genotype
Acknowledgements
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The Howard Hughes Medical Institute
Dr. John Hays
Dr. Marc Curtis
Buck Wilcox
Peter Hoffman
Shauna Tominey
Dr. Kevin Ahern