Recruitment and activation of the ATM kinase in the absence of … · 2016. 5. 10. · Ferguson Lab 12 May 2015. The Mre11/Rad50/NBS1 complex (MRN) Rad50 Rad50 Kanaar and Wyman (2008)

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


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



Endrecognition

ATM activation

Checkpointinduction

Senescence, apoptosis


MRN Ku70/80

End bindingor synapsis

KuDNA-PKcs


MRN and ATM control the G2/M cell cycle checkpoint



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


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

− +


− + − +

+/− −/− −/−+/+ +/+ −/−

− +


− + − +

+/− −/− −/−+/+ +/+ −/−

− +


− + − +

+/− −/− −/−+/+ +/+ −/−

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

− +


− + − +

+/− −/− −/−+/+ +/+ −/−

− +


− + − +

+/− −/− −/−+/+ +/+ −/−

− +


− + − +

+/− −/− −/−+/+ +/+ −/−

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


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


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


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


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

Documents

Recruitment and activation of the ATM kinase in the absence of … · 2016. 5. 10. · Ferguson Lab 12 May 2015. The Mre11/Rad50/NBS1 complex (MRN) Rad50 Rad50 Kanaar and Wyman (2008)