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Recruitment and activation of the ATM kinasein the absence of DNA damage sensors
Andrea Hartlerode PhDUniversity of Michigan
Ferguson Lab12 May 2015
The Mre11/Rad50/NBS1 complex (MRN)
Rad50 Rad50
Kanaar and Wyman (2008) Cell 135:14-6
Mre11 Nuclease domain
1− −708
Exon 5Mre11+
Mre11cond
Mre11−
Cre recombinase
Exon 5Phenocopieswildtype allele
Embryonic lethal(homozygous)
The Mre11/Rad50/NBS1 complex (MRN)
Rad50 Rad50
Kanaar and Wyman (2008) Cell 135:14-6
Mre11 Nuclease domain
1− −708
Mre11cond/− to Mre11−/−
Mre11
Rad50
NBS1
Tubulin
+/− P0 P1 P2 P3
passage post-Cre
Activation of the ATM kinase is disruptedin Mre11-deficient cells
Rad50 Rad50
ATMATM
ATM
P
Inactivekinase
Activekinase
Kanaar and Wyman (2008) Cell 135:14-6Buis et al. (2008) Cell 135:85-96
−
Mre11+/+
+
Mre11−/−
− +
pSMC1S957
α-Tubulin
10Gy
The ATM kinase controls DSB responses
adapted from Stracker and Petrini (2011) Nat Rev Mol Cell Bio 12:90-103
Double-strandbreak (DSB) induction
Endrecognition
ATM activation
Checkpointinduction
Senescence, apoptosis
DNA double-strand break repair
MRN
Ku DSB sensor recruits/activates the kinase DNA-PKcs
adapted from Stracker and Petrini (2011) Nat Rev Mol Cell Bio 12:90-103
Double-strandbreak (DSB) induction
Endrecognition
ATM activation
Checkpointinduction
Senescence, apoptosis
DNA double-strand break repair
MRN Ku70/80
End bindingor synapsis
KuDNA-PKcs
DNA double-strand break repair
MRN and ATM control the G2/M cell cycle checkpoint
adapted from Stracker and Petrini (2011) Nat Rev Mol Cell Bio 12:90-103
Double-strandbreak (DSB) induction
Endrecognition
ATM activation
Checkpointinduction
MRN
Cell cycle
G1
SG2
M
G1 checkpoint
Intra-S checkpoint
G2/M checkpoint
Spindleassemblycheckpoint
(pH3S10 +)
ATM-/- Mre11+/-
Ku70+/+Mre11-/-
Ku70+/+
0.0
0.2
0.4
0.6
0.8
1.0
Mito
tic In
dex
(+IR
/-IR
)
Loss of the MRN complex impairs the G2/M checkpoint
Buis et al. (2008) Cell 135:85-96
Mito
tic In
dex
Rat
io (+
IR/−
IR)
ATM-/- Mre11+/-
Ku70+/+Mre11-/-
Ku70+/+Mre11+/-
Ku70-/-Mre11-/-
Ku70-/-
0.0
0.2
0.4
0.6
0.8
1.0
Mito
tic In
dex
(+IR
/-IR
)
The defective G2/M checkpoint in MRN-deficient cellsis rescued by Ku deficiency
Mito
tic In
dex
Rat
io (+
IR/−
IR)
Mre11+/-
Ku70+/+Mre11-/-
Ku70-/-
0.0
0.2
0.4
0.6
0.8
1.0
Mito
tic In
dex
(+IR
/-IR
)No inhibitorATMiPKi
The rescued G2/M checkpoint in MRN/Ku-deficientcells is ATM-dependent
PKi = DNA-PK inhibitor (NU7026)ATMi or Ai = ATM inhibitor (KU55933)
Mito
tic In
dex
Rat
io (+
IR/−
IR)
MRN-dependent recruitment of ATM to chromatin
Double-strandbreak (DSB) induction
Endrecognition
ATM activation
Checkpointinduction
MRN
ATM chromatinlocalization
ATM localizes to chromatin following DNA damage
GAPDHH2AX
ATMγH2AX
IR
Mre11Ku70
− +
Fraction I (cytoplasmic)
− + − +
+/− −/− −/−+/+ +/+ −/−
− +
Fraction II (nuclear)
− + − +
+/− −/− −/−+/+ +/+ −/−
− +
Fraction III (chromatin)
− + − +
+/− −/− −/−+/+ +/+ −/−
Andegeko et al. (2001) J Biol Chem 246:38224-30
ATM cannot localize to chromatin in the absence of MRN
GAPDHH2AX
ATMγH2AX
IR
Mre11Ku70
− +
Fraction I (cytoplasmic)
− + − +
+/− −/− −/−+/+ +/+ −/−
− +
Fraction II (nuclear)
− + − +
+/− −/− −/−+/+ +/+ −/−
− +
Fraction III (chromatin)
− + − +
+/− −/− −/−+/+ +/+ −/−
Uziel et al. (2003) EMBO J 22:5612-21
Ku removal restores damage-induced ATM chromatin recruitment in the absence of the MRN complex
GAPDHH2AX
ATMγH2AX
IR
Mre11Ku70
− +
Fraction I (cytoplasmic)
− + − +
+/− −/− −/−+/+ +/+ −/−
− +
Fraction II (nuclear)
− + − +
+/− −/− −/−+/+ +/+ −/−
− +
Fraction III (chromatin)
− + − +
+/− −/− −/−+/+ +/+ −/−
ATM-dependent nuclear phosphorylation events are rescued by loss of Ku in MRN deficiency
pKap1S824− A
iP
Ki
10Gy
− AiP
Ki
pSMC1S957
GAPDH
− Ai
PK
i
10Gy
− AiP
Ki
− Ai
PK
i
10Gy
− AiP
Ki
− Ai
PK
i
10Gy
− AiP
Ki
PKi = DNA-PK inhibitor (NU7026)ATMi or Ai = ATM inhibitor (KU55933)
Mre11+/−Ku70+/+
Mre11−/−Ku70+/+
Mre11+/−Ku70−/−
Mre11−/−Ku70−/−
Mre11c/-
Ku70+/+Mre11-/-
Ku70+/+Mre11c/-
Ku70-/-Mre11-/-
Ku70-/-
0.0
0.2
0.4
0.6
0.8
1.0
Rel
ativ
e H
R F
requ
ency
Normalized
Ku deficiency does not rescue MRN-dependenthomologous recombination (DR-GFP)
Normalized
no rescue
The role of MRN may not be as simple asdirect recruitment and allosteric activation of ATM
Rad50 Rad50
ATMATM
ATM
P
Inactivekinase
Activekinase
Kanaar and Wyman (2008) Cell 135:14-6
In the absence of MRN:Ku removal restores:• ATM chromatin
localization• ATM kinase activation• The ATM-dependent
G2/M cell cycle checkpoint
Ku removal does not rescue:• MRN-dependent HR
DNA-PKcs can efficiently phosphorylate H2AXin the absence of MRN
− − Ai AiPKiPKi
3 Gy
Mary Morgan
PKi = DNA-PK inhibitor (NU7026)ATMi or Ai = ATM inhibitor (KU55933)
γH2AX
DAPI
γH2AX
DAPI
Mre11+/−Ku70+/+
Mre11−/−Ku70+/+
γH2AX foci in the absence of Mre11 and Ku areATM-dependent and indistinguishable from controls
Mary Morgan
γH2AX
DAPI
γH2AX
DAPI
PKi = DNA-PK inhibitor (NU7026)ATMi or Ai = ATM inhibitor (KU55933)
Mre11+/−Ku70+/+
Mre11−/−Ku70−/−
− − Ai AiPKiPKi
3 Gy
Antagonism between Mre11 and Ku at some level?
Competition for DNA ends?
P Ku70/80
H2AX H2AX H2AX H2AX H2AX
MRN
H2AX
P
H2AXKu70/80
H2AX H2AX H2AX
Opposing functions after both sensors are recruited?
MRN
MRN recruitment is not altered by loss of Ku
30 900 15 60 120Time after microirradiation (seconds)
Ku70+/+
Ku70−/−
5
NBS1-GFP
Ku recruitment is rapid and unaltered by loss of MRN
GFP-Ku70/80
Summary
In the absence of MRN, Ku removal restores:• ATM chromatin localization• ATM kinase activation• The ATM-dependent G2/M cell cycle
checkpoint
In the absence of MRN, Ku removal does not restore:
• MRN-dependent homologous recombination
Implications
ATM
H2AX H2AX H2AX H2AX
P
H2AX
MRN
MRN
Not essentialfor ATM activation
Optimization of ATMactivation and signaling
Ku70/80
MRN may oppose Ku
Acknowledgements
Ferguson Lab (Univ. of Michigan)David Ferguson MD/PhDHilary MoaleMary MorganJeff Buis PhDTodd Festerling PhDJosh Regal PhDElizabeth Spehalski PhDYipin Wu PhD
Yu Lab (Univ. of Michigan)Xiaochun Yu PhDChao Liu PhDLin-Yu (Charles) Lu PhD
Our NeighborsSekiguchi LabCanman Lab
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