Roles of Mammalian Mre11 in Genomic Stability and Development

Roles of Mammalian Mre11 in genomic stability and development

David Ferguson MD, PhD Department of Pathology University of Michigan Medical School The Mre11/Rad50/NBS complex (MRN)

Kanaar & Wyman, Cell 2008 The MRN complex: a sensor of double strand breaks

R R

DSB M M N

P ATM ATM ATM Activation

DNA Repair Cell cycle checkpoints

Arrest or apoptosis ATM and MRN mutations compared • Human Syndromes – Many AT patients harbor ATM null mutations. – All ATLD and NBS patients have hypomorphic Mre11 or NBS1 mutations.

• Mouse knockouts – ATM knockout mice are viable. – MRN knockouts are very early embryonic lethal (RN). The MRN complex: a sensor of double strand breaks

R R

DSB M M N P ? P ATM ATM ATM Activation

DNA Repair Cell cycle checkpoints

Arrest or apoptosis The Mre11/Rad50/NBS complex (MRN)

Ataxia telangiectasia like disorder (ATLD)

(Nijmegen Breakage Syndrome) The Mre11 nuclease - in vitro activities

Substrate + Rad50 + NBS In vivo role? or XRS 5' 3' 3' recessed + + expose microhomology? Blunt + + }

ds exonuclease 3' overhang - ND Prepare for NHEJ? 5' 3' 3' overhang Prepare for NHEJ? 5' overhang - ND Prepare for NHEJ?

3' ssDNA ﬂap - ND Restore replication fork? 3' ssDNA branch + ND } ss endonuclease hairpin with loop + + V(D)J recombination? hairpin w/o loop + + } Closed ssDNA + ND Generation of a mouse Mre11H129N nuclease deﬁcient allele

MmMre11

CAT

AAT N = Mre11H129N In Saccharomyces cerevisiae, point mutations in the Mre11 nuclease domains confer variable ionizing radiation hypersensitivity

Wild type

Nuclease mutations IV *

III Nuclease domains

II I Null N

Krogh, Symington, Genetics, December 2005 Mutation of human Mre11 nuclease motif III disables exonuclease activity

(Bressan et al. Genetics 1998)

(Arthur et al., NAR 2004) Mutation of Mre11 aa H85 (domain III) in P. furiosus abrogates nuclease activity, no change to crystal structure

N * III Generation of a mouse Mre11H129N nuclease deﬁcient allele

MmMre11

CAT

AAT Why H129N? N = Mre11H129N • 100% conserved in evolution. • Histidine129 required for transesteriﬁcation reaction. • Similar mutations abrogate nuclease activity in vitro in yeast and human. • NOT required for structure or protein-protein interactions. • Same mutation in yeast causes mild IR sensitivity, blocked meiosis. Mre11H129N/H129N causes lethality early in development: No rescue by p53 deﬁciency

Mre11 genotype p53 genotype LIVE BORN +/+ +/- -/- H129N/WT H129N/H129N + / + 19 24 16 + / H129 27 37 21 H129N / H129N 0 0 0 e9.5 DAY E13.5 +/+ +/- -/- + / + 5 13 9 + / H129 18 24 12 H129N / H129N 0 0 0

DAY E9.5 +/+ +/- -/- + / + 5 13 6 e7.5 + / H129 7 14 11 H129N / H129N 2 5 4 Three germline alleles of mouse Mre11 1. Mre11 nuclease deﬁcient allele (H129N)

MmMre11

CAT AAT N 2. Mre11 conditional allele = Mre11H129N LoxP LoxP Mre11cond

Cre recombinase 3. Mre11 null allele LoxP Mre11null Four germline mouse alleles of Mre11

H H Mre11cond A

LoxP LoxP D

H H Mre11Δ A Mre11 cond / Δ D LoxP

H H H WT cond / WT Mre11 A Mre11

X H129N H H H Mre11 A Mre11 cond / H129N LoxP D Cre delivered via adenovirus mediates conversion of Mre11Cond to Mre11Δ in mouse embryonic ﬁbroblasts (MEFs)

Cond/wt Δ/wt Cond/Δ Δ/Δ Cond/H129N Δ/H129N P0 P1 P2 P10 P0 P1 P2 P10 P0 P1 P2 P10 Mre11 Rad50 NBS1 Tubulin Mre11H129N does not disrupt the MRN complex

A. Co-IP of endogenous proteins. B. Two Hybrid: Mre11H129N homodimerizes.

Genotype +/Δ H129N/Δ Δ /Δ +/Δ Mre11 + + + - α Prey Bait 5000 500 50 cells Rad50 Mre 11 Mre 11 NBS1 Mre 11129N Mre 11 129N Mre11

Mre 11 no insert 1.4 +/∆ 1.2 Mre11 H129N/∆ no insert Mre 11 1 Mre11 0.8 0.6 0.4

0.2Ratio relative to Mre11 0 Mre11 Rad50 NBS1 The Mre11H129N protein can form IR induced foci Implication: Nuclease deﬁciency does not impair localization to DSBs

Mre11+/Δ Mre11Δ/Δ Mre11H129N/Δ

No ionizing radiation

αMre11

+ ionizing radiation Severe proliferation defect in primary MEFs conferred by MRN loss or Mre11 nuclease deﬁciency

2500000 Mre11 +/∆ Mre11 ∆/∆ Mre11 H129/∆

2000000

1500000

1000000 Total number of cells Total 500000

0 1 2 3 4 5 Passage Signiﬁcant rescue of proliferation defects by large T antigen immortalization

10000000 Mre11 +/∆ 9000000 Mre11 ∆/∆ Mre11 H129N/∆

8000000

7000000

6000000 5000000

4000000 3000000 Total number of cells Total 2000000

1000000 0 1 2 3 4 5 6 7 8 9 10 Passage Proliferation defects are associated with premature senescence (β-gal staining)

60 P2 primary MEFs 50 P3 primary MEFs P3 transformed MEFs * 40

20 % positively stained 10

0 * * * +/∆ ∆/∆ H129N/∆ Ku -/- Mre11 Genotype Dramatic chromosome instability in Mre11 null MEFs

Translocations (SKY) - Chromatid gap

- Radial

Fragment - Genomic instability: loss of MRN versus Mre11 nuclease 3.5 Radials 3 Radial Fusions

2.5 Gaps/Breaks Fragments Fusion 2

1.5

1 Anomalies per metaphase 0.5 Fragment

0 P T P T P T P P-AdE T

+/Δ Δ/Δ H129N/Δ +/+ Cond/Δ Mre11 Lig4-/- • Mre11 nuclease deficiency phenocopies loss of entire MRN complex. • Genomic instability not impacted by cellular transformation. • Genomic instability conferred by Mre11 and Lig4 deficiencies are distinct; - Mre11 deficiencies more severe and feature gaps, radials, fusions. Mre11Δ/Δ and Mre11H129N/Δ confer equivalent hypersensitivity to IR

100

% Survival Mre11 +/∆ Mre11∆/∆ Mre11 H129N/∆ Lig4-/-

1 0 200 400 600 800 1000 Dose (Rads) Does the nuclease deﬁcient MH129NRN complex activate ATM?

R R DSB M M N P ? P ATM ATM ATM Activation

DNA Repair Cell cycle checkpoints

Arrest or apoptosis Mre11 nuclease deﬁciency does not impact ATM activation induced by ionizing radiation

Mre11+/Δ Mre11Δ/Δ Mre11H129N/Δ

IR - + - + - + pATMser1987

pCHK2 CHK2

pSMC1ser957

α-tubulin Intact G2/M checkpoint in Mre11 nuclease deﬁciency (staining for phospho-histone H3)

1.4 Mock IR 1.2 1.0 0.8 0.6 0.4 0.2

Relative % p-H3 pos 0.0 WT/WTΔ ΔD/Δ H129N/H129NΔ Nuclease activities of mammalian Mre11 provide an essential function distinct from MRN control of IR induced ATM activation

Next question: Do our observations hold in the absence of ionizing radiation? ATM activation induced by dysfunctional telomeres depends on the MRN complex, but not Mre11 nuclease activity (transfection of dominant negative TPP1 allele)

Mre11Δ /+ Mre11Δ / Δ Mre11Δ / H129N TPP1RD - + - + - + - + - + - + Cre - - + + - - + + - - + +

p-ATM

ATM

TPP1∆RD Fusions in Mre11 deﬁciencies do not involve telomeres

- MRN is not a Shelterin component -

Mre11Δ/Δ or Mre11H129N/Δ TPP1acd/acd Mre11Δ/Δ

Mre11H129N/Δ What are the nuclease activities of Mre11 doing?

R R

DSBM M N Mre11 nuclease activities ?

P ATM ATM ATM Activation

DNA Repair Cell cycle checkpoints

Arrest or apoptosis Persistence of chromosome anomalies after IR - suggesting a severe DSB repair defect? -

12 Fusions 10 Gaps/Breaks 8 Fragments

anomalies per metaphase 2

0 24 48 72 24 48 72 24 48 72 24 48 72 Mre11+/Δ Mre11Δ/Δ Mre11 H129N/Δ Lig4-/- Pulsed ﬁeld gel DNA repair assay; Absence of MRN or Mre11 nuclease activities cause defective double strand break repair Mre11 +/Δ Lig4 -/- Mre11 Δ /Δ Mre11H129N/Δ 1 ...... 18 1 ...... 18 1 ...... 18 1 ...... 18 Hours post IR

1.2

0.8

0.6 Mre11+/∆ Mre11∆/∆ 0.4 Mre11H129N/∆ Lig4-/- 0.2 DSB level (fraction of initial damage) 0 0 1 2 4 6 18 Time (Hr) Does mammalian MRN function in NHEJ? V(D)JV(D)J Recombination recombination Assay - transient transfection assay

V(D)J Recombination Substrate Signal Join substrate Coding Join substrate

STOP STOP

RAG1 RAG2 RAG1/2 RAG1/2

Embryonic Blunt Hairpins Fibroblasts Ku70 Artemis Ku80 DNA-PK Lig4 Ku70/80 Recombined product XRCC4 XLF Lig4 Bacterial XRCC4 Transformation Signal XLF & Selection Join

Coding Join All plasmids Recombined plasmids Mre11 and non-homologous end joining; No impact of Mre11 deﬁciency on plasmid based V(D)J recombination

Signal joins - blunt ends Coding joins - hairpin ends

1.2 1.8 Signal Joint - 1 1.6 Coding Joint - 1 1 Signal Joint -2 Coding Joint - 2 1.4

0.8 1.2

1 0.6 0.8

0.4 0.6

0.4 0.2 0.2 Relative Recombination Frequency

Relative Recombination Frequency 0 0 +/∆ No RAG +/∆ ∆/∆ H129N/∆ Lig 4 -/- +/∆ No RAG +/∆ ∆/∆ H129N/∆ Lig 4 -/- Mre11 nuclease activities and homologous recombination?

5' 3' Resection of single strand (Mre11 nuclease activities?)

5' 3' Bound by RPA (SSB) 5' 3' Rad51 ﬁlament 5' 3' Homologous recombination

3'5' 3' Reduced RPA focus formation in MRN or Mre11 nuclease deﬁciency

12 10 8 Mre11+/Δ Mre11 Δ/Δ Mre11 H129N/Δ 6 4

Foci per cell 2 0 0 Gy non-ir ir non-ir ir non-ir ir +/∆ ∆/∆ H129N/∆

70 10 Gy 60 50 40 30 20 10

% cells with >10 foci 0 non-ir ir non-ir ir non-ir ir +/∆ ∆/∆ H129N/∆ Reduced Rad51 focus formation in MRN or Mre11 nuclease deﬁciency

+/Δ Δ/Δ H129N/Δ Mre11 Mre11 Mre11 80% 70% 60% 0 Gy 50% 40% 30% 20% 10% % cells with >10 foci 10 Gy 0% non-IR IR non-IR IR non-IR IR

+/∆ ∆/∆ H129N/∆ Direct assessment of homology directed repair

GFP-

Homology directed repair + GFP GFP

(Nakanishi et al., 2005) Dramatic reduction in homology directed repair conferred by deﬁciency of MRN or of Mre11 nuclease activities

GFP-

Homology directed repair + GFP GFP

(Nakanishi et al., 2005)

120% • Average of three independent DR Ad I-Sce 100% -GFP lines for each genotype. Ad E 80% • +/Δ control set to 100%. 60%

40% • Mre11Δ/Δ and Mre11H129N/Δ are

20% each <10% of control. Relative HDR frequency 0% +/∆ ∆/∆ H129N/∆ Mre11 nuclease activities and ATR activation?

5' 3' Resection of single strand (Mre11 nuclease activities?)

5' 3' Bound by RPA (SSB) 5' 3'

ATR ATR 5'ATRIP ATRIP 3' P Chk1 UV induced Chk1ser345 phosphorylation depends on Mre11 nuclease activities; implication - role in ATR activation

No UV 10 min 30 min Recovery time after 2 + ∆ H129N + ∆ H129N + ∆ H129N low dose UV (5 J/m ) pChk1-ser345 GAPDH The nuclease activity of Mre11 is required during replication stress

100 14 Radials 12 Fusions 10 Gaps/Breaks Fragments 8 10 6 Mre11+/Δ Mre11Δ/Mre11Δ H129N/Lig4-/-Δ 4 2 anomalies per metaphase

% cell survival 0 1 0 72 0 72 0 72 0 72 0 0.5 1 1.5 2 +/Δ Δ/Δ H129N/Δ Lig4-/- Mre11 +/∆ Hours after 24 hr exposure (1 uM) Mre11 ∆/∆ Mre11 H129N/∆ Lig4-/- 0.1 Dicentrics in 25 metaphases Aphidicolin (uM) 4 46 39 9 Summary In mammals, the nuclease activities of Mre11 are; essential in early mammalian development. fundamentally important for maintaining genomic stability, and resistance to IR and replication stress (aphidicolin). play an important role in DSB repair via homologous recombination, likely during resection. contribute to ATR activation (likely through ss DNA) The essential role(s) is distinct from MRN control of ATM activation. ATP dependant unwinding of single strands by Rad50 is separable from ATP dependant resection by Mre11. Mre11 Nuclease Activities and ATM control are separable functions of the MRN complex

Long range interactions (Rad50)

substrates Nucleolytic processing by Mre11 – HDR (and some ATR activation). Initiates HDR by generating a short 3' overhang – Other nucleases and helicases Active generate long 3' overhang. kinase P- ATM

Short range interactions (Mre11 dimer). Inactive DNA unwinding (Rad50). kinase ATM