Mitochondrial DNA Mutations Cause Various Diseases

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2013 Neurobiology of Disease in Children Symposium: Mitochondrial Disease, October 30, 2013 Defects of Mitochondrial DNA Replication

William C. Copeland Laboratory of Molecular Genetics Mitochondrial DNA mutations cause various diseases

* Alpers Disease * Leigh Disease or Syndrome * Barth syndrome * LHON * Beta-oxidation Defects * LIC (Lethal Infantile Cardiomyopathy) * Carnitine-Acyl-Carnitine * Luft Disease Deficiency * MAD * Carnitine Deficiency * MCAD * Co-Enzyme Q10 Deficiency * MELAS * Complex I Deficiency * MERRF * Complex II Deficiency * Mitochondrial Cytopathy * Complex III Deficiency * Mitochondrial DNA Depletion * Complex IV Deficiency * Mitochondrial Encephalopathy * Complex V Deficiency * Mitochondrial Myopathy * COX Deficiency * MNGIE * CPEO * NARP * CPT I Deficiency * Pearson Syndrome * CPT II Deficiency * Pyruvate Carboxylase Deficiency * Glutaric Aciduria Type II * Pyruvate Dehydrogenase Deficiency * KSS * Respiratory Chain * Lactic Acidosis * SCAD * LCAD * SCHAD * LCHAD * VLCAD Origins of mtDNA mutations

Damage to DNA •Environmental factors •Endogenous oxidative stress

Spontaneous errors •DNA replication •Translesion synthesis •DNA repair re-synthesis Mitochondrial DNA replication

p32 - RNaseH 16 Human DNA Polymerases

Polymerase Family Chromosome Mol. Wt. (kDa) Function/Comments

α (alpha) B Xq21.3-q22.1 165 Initiates replication

β (beta) X 8p12-p11 39 BER, other functions

γ (gamma) A 15q25 140 Mitochondrial replication & repair

δ (delta) B 19q13.3-.4 125 Replication, BER, NER, MMR

ε (epsilon) B 12q24.3 255 Replication, checkpoint control

ζ (zeta) B 6q22 344 yREV3 homolog, lesion bypass

η (eta) Y 6p21.1 78 Lesion bypass, XPV, skin cancer susceptibility

θ (theta) A 3q13.31 300 crosslink repair, Dm308, lesion bypass

ι (iota) Y 18q21.1 80 Lesion bypass? BER?

κ (kappa) Y 5q13.1 99 Lesion bypass, mutator when overexpressed

λ (lambda) X 10q23 64 pol β homolog, meiosis? NHEJ

µ (mu) X 7p13 55 TdT homolog, NHEJ

n (nu) A 4p16.3 100 lesion bypass, crosslink repair?

σ (sigma) X 5p15 60 TRF4

Rev1 Y 2q11.1-.2 125 lesion bypass TdT X 10q23-24 57 Terminal transferase Human DNA Polymerase γ

Pol γ is a trimeric complex composed of two gene products: 140 kDa and two p55 kDa polypeptides

POLG encoding p140: Catalytic subunit • DNA polymerase activity • 3’-5’ exonuclease activity • 5’-dRP lyase activity

POLG2 encoding p55 dimer: Accessory subunit • Processivity factor • DNA binding factor p32 - RNaseH

The p55 accessory subunit increases the processivity of the DNA polymerase by several hundred fold, stimulates DNA synthesis, and restores salt-tolerance through enhanced DNA binding. DNA polymerase γ participates in both mitochondrial DNA replication and repair. ~90% of mutations detected in vivo are reproduced in vitro with recombinant DNA polymerase γ Proteins involved in mitochondrial DNA replication

5 '

mtSSB p140 - POLG

mtSSB p72 - Helicase

p55 - POLG2 mtSSB

p32 - RNaseH Polγ Twinkle 5 ' 3 ' Helicase 5 ' 3 ' p55 p15 - mtSSB

NDP RRM2B TP TK1 thymine thymidine dTMP dTDP dTTP

DNC

SUCLA2 DNA replication SUCLG1 MGME1 TK2 NDPK PEO1 thymidine dTMP dTDP dTTP POLG2 deoxycytidine dCTP dCDP dCTP dGuoK POLG deoxyguanine dGMP dGDP dGTP MtDNA deoxyadenine dAMP dADP NDPK dATP SUCLA2 OXPHOS SUCLG1 ADP ATP OPA1 MPV17? ANT1

ADP ATP

Copeland, Ann. Rev. Med 2008 Human POLG gene 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Mitochondria targeting sequence I II III A B C Exonuclease Thumb Spacer Thumb Palm Fingers Palm

25 170 440 475 785 855 910 1104 1239

p55 binding

Exonuclease domain Linker region Polymerase domain Crystal structure of the human pol γ holoenzyme Human POLG gene 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Exonuclease domain Linker region Polymerase domain

Mitochondria targeting sequence I II III A B C Exonuclease Thumb Spacer Thumb Palm Fingers Palm

25 170 440 475 785 855 910 1104 1239

p55 binding

R3P L304R A467T Y955C

2001 Van Goethem et al., Nat. Genet. Mutation of POLG is associated with Progressive External Ophthalmoplegia characterized by mtDNA deletions Mutations in DNA polymerase γ, POLG Human POLG gene 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Q879H E873X E1136K R869Q T885S A767D H1134R E1143G* H277C L304R L304R G517V K561M A862T L886P T251I S305R G763R G888S H1110Y E1143G* Q497H R853Q L244P H569Q L752P E895G R309H A467T R1096H 3482+2(T>C)splice R574W F749S R852C T914P R1096C W235X T326fs387X A467T M1163R P587L T851A K925fsX R1047W R232G P648R W748S R1187W L428P T849X D930N R232H P589L W748S W1020X L83P R943C L1173fs X R227P W347fs356X L605R G848S A957P G11D Q68X R227W G737R K1191N R374X R627W 2485∆12bp R964C G11D 2157 splice R964C K1191R C224Y C418R R627Q 2480 splice Mitochondria A143V R417T Q715X R807C L965X Y1210fs1216X R627Q L966R 3643 splice targeting sequence I II III A B C Exonuclease Thumb Spacer Thumb Palm Fingers Palm F961S R3P L424X L623W A804T R227W R807P A957S G1205A Q43R G426S R617C P648R T251I Y831C* Y955C D1196N Q45R M430L M603L R953C G268A R709X G848S D1184N ∆(CAG)n G431V R597W R1047W R1187W R275X G746S R853W R1047Q S433C S1176L H110Y L304R P587L V855A R943H G1051R T452X K755E F1164I A862T Q308H R579W H932Y G1076V R1138C D136E L463F G763R N864S R309L R574W G923D I1079L R1128H A467T 2354Gins A889T V1106I W312R R562Q W918R S1095R G380D N468D A1105T S511N R1096C S1104C Exonuclease domain Polymerase domain - adPEO+ (Progressive External Ophthalmoplegia) -Alpers and other Infantile Hepatocerebral Syndromes - NRTI toxicity with mtDNA depletion - arPEO+ -Male infertility / - Ataxia-Neuropathy Syndrome, MIRAS / SANDO / SCAE - PEO, sporadic - testicular cancer / Idiopathic Parkinson - Single Nucleotide Polymorphism (*) - Other http://tools.niehs.nih.gov/polg/ Major clinical syndromes associated with POLG mutations

Age of Onset Syndrome Myocerebrohepatopathy spectrum Neonatal/Infancy (MCHS) Alpers-Huttenlocher syndrome Infancy/Childhood (AHS)

Ataxia neuropathy spectrum (ANS)

Myoclonus, epilepsy, myopathy,

Adolescent/young adult sensory ataxia (MEMSA) Progressive external ophthalmoplegia (PEO) with or without sensory ataxic neuropathy and dysarthria (SANDO) Mutations in DNA polymerase γ, POLG Human POLG gene 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Easily screen for mitochondrial Conserved regions with human POLG dysfunction

Yeast genetics

Heteroallelic Monoallelic Strand et al., 20030

WT MIP1 ΔWT MIP1 X Assay for mtDNA mutants

mut mip1 mut mip1 • mtDNA depletion • mtDNA point mutations • mtDNA deletions

Monoallelic mutants that increase petite colony formation frequency Table 2. Monoallelic mip1 mutations that increased petite frequency Genotypea % Petite Wildtype 10 Exo- 46 L260R 40 Q264H 100 R265L 35 R607C 100 R607P 67 G651S 100 T654A 100 R656W 100 R656Q 100 N667S 100 G691S 18 H734Y 100 A759P 100 G832V 100 R853C 100 R853H 100 S861C 100 V863I 100 D941N 33 a All strains contain plasmid pFL39 with a mutation causing the indicated amino acid change and contain chromosomal mip1 deletion Stumpf et al. HMG 2010,

What causes mitochondrial dysfunction in mip1 mutants?

• MtDNA depletion • Point mutagenesis • Deletions mip1 disease mutations increase mtDNA mutagenesis

(heteroallelic: wt/mut)

Stumpf et al. HMG 2010, Homozygous POLG exo-/- mice have premature aging phenotypes But POLG exo+/- heterozygous mice appear normal

+/- exo exo-/-

Homozygous POLG exo-/- mice display a 2000-fold increase in point mutations Heterozygous POLG exo+/- display a 500-fold increase in point mutations

Trifunovic et al., 2004; Kujoth et al., 2005 Vermulst et al. 2007 Exonuclease domain mip1 mutations cause very little increase in mutant frequency

Heteroallelic strains Monoallelic strains

600 80

70 500

60 400

300 40

30 200

20 100 10

0 0 Mutant Frequency relative to wild-type Mutant Frequency Relative to wildtype wild- exo- G224A L210P L260R Q264H R265L R265H wild- exo G224D G224A L210P L260R R265L R265H type type

Stumpf et al., 2010, Hu et al., 1995, Baruffini et al., 2006 What about deletions?

• What is the role of Pol γ exonuclease in mtDNA deletions? POLG disease variants do not show significant deletion mutagenesis

Deletion mutagenesis in heterozygous strains

Phadnis, Sia and Sia, 2005 Genetics

Stumpf and Copeland 2013 Genetics The Pol γ exonuclease suppresses the formation of deletions between direct repeats in mtDNA

Stumpf and Copeland 2013 Genetics Summary of POLG studies

• Most disease mutations in POLG led to loss of mitochondrial DNA with loss of respiratory activity

• Dominant mutations map to the polymerase domain

• Increased point mutagenesis in POLG related disorders plays a limited role in mitochondrial disease and aging

• Disease mutations in the exonuclease affect DNA polymerization but not the exonuclease activity.

• The POLG exonuclease suppresses mtDNA deletions POLG2, the accessory subunit of Pol γ (p55 dimer, functions as processivity factor)

Research focus:

• Animal model

• Study of POLG2 disease mutation p55 is Absent in Less Complex Eukaryotes

• Single cell eukaryotes through nematodes lack an accessory subunit • The insect accessory subunit is as a monomer • Vertebrates accessory subunit is a homodimer POLG2 Mouse Knockout

Genotypes of live pups from crosses of POLG2+/- x POLG2+/-

Genotype Count Percent POLG2 HET (+/-) 170 68% WT (+/+) 81 32% POLG2 KO (-/-) 0 0% TOTAL: 251

*Data comprised of 40 litters with an average of 6 pups per litter

POLG2 -/- is embryonic lethal The POLG2 -/- embryo arrests at embryonic day 8.5

POLG2-/- embryo has morphology of an E8.5 embryo POLG2 is an Essential Gene

A. Histochemical staining of COX (a mitochondrial encoded gene product) and Hematoxylin reveals the absence of COX in POLG2 -/- embryos

B. Real-time PCR quantification of mouse mitochondrial DNA demonstrates nearly complete loss of mtDNA in POLG2 -/- embryos but near normal levels in POLG2 +/- heterozygotes EM reveals ultra-structural defects with loss of cristae, swollen mitochondrial and accumulated lipid droplets Wild Type POLG2 KO

11,500X 20,500X Homozygous POLG2 KO embryonic tissue at 43,000X Summary of POLG2 mouse KO model:

• POLG2 knockout mouse is embryonic lethal • Arrested development at day 8.5 p.c. • Determined homozygous KO’s have depleted mtDNA and no COX activity • EM analysis of homozygous KO heart embryos show ultrastructural abnormalities and loss of cristae • Heterozygous POLG2 KO’s appear to be asymptomatic up through 2 years of life • Heterozygous POLG2 KO’s have normal mtDNA levels and histopathology G103S

L153V

P205R

R369G D386E

G416A S423Y G451E

L475DfsX2 Longley et al., AJHG 2006 Young et al., HMG, 2011 Craig et al., Mitochondrion 2012 Processivity and Activity of Pol γ with the POLG2 variants p140+p55

p140 alone

Young et al., HMG, 2011 Purification of p55 heterodimers for in vitro biochemical studies G451E-p55 is Dominant Negative In Vitro

Alkaline agarose gel displaying DNA synthesis of either the WT p140 or exonuclease deficient 140 with heterodimeric disease variants of p55 Cell Models of POLG2 Disease

A mammalian expression vector was constructed to examine the expression and subcellular localization of wild-type p55 and disease variants in HEK-293 cells. Localization of p55 variants

A. WT B. C. D.

E. NoMTS F. G. H.

I. P205R J. K. L. Quantifying the Failure to Form Puncta

Indicator of intramitochondrial mistargeting POLG2 mutations cause mtDNA deletions in cell lines

R369G-POLG2 causes mtDNA deletions in tissue culture cells Biochemistry summary of POLG2 variants

Homodimers: • G103S, L153V, D386E and S423Y proteins display wild-type behavior. • P205R and R369G p55 variants has reduced stimulation of processivity and decreased affinity for the catalytic subunit. • L475DfsX2 does not bind p140 catalytic subunit nor bind dsDNA and forms aberrant oligomeric complexes. • G451E p55 is unable to bind the p140 catalytic subunit

Heterodimers: • All heterodimers bind p140 • G451E is dominant negative

Cell culture: • P205R and L457fsX2 do not localize to nucleoids • R369G causes increased mtDNA deletions Matt Longley Maggie Humble

Matt Young Sam Gattis