Mitochondrial DNA and Non-Mendelian Inheritance

Carolyn K. Suzuki, Ph.D.

Dept of Biochemistry
 Essential functions of mitochondria

Biosynthesis of
amino acids
nucleotides
steroid hormones
heme
ATP synthesis
Oxidation of fatty acids
Apoptotic cell death
Mitochondrial proteins are encoded by 2 separate genomes and
translated by 2 different protein synthesis machineries
Proteins localized in mitochondria are:

encoded by two genomes-

•nuclear DNA (nDNA)
•mitochondrial DNA (mtDNA)

produced by two different protein synthesis machineries

•cytosolic
•mitochondrial

majority of mitochondrial proteins are encoded by nuclear

genes, which are synthesized in cytosol and post-translationally
imported into mitochondria.

13 mitochondrial proteins are encoded by mtDNA, which are

synthesized in the mitochondrial matrix.
 Organization of the mitochondrial genome
D-loop
non-coding region

16,659 bp

tRNAs
D-loop: displacement loop
HSP and LSP: heavy- and light- strand promoters for transcription
OH: origin of replication
Characteristics of animal mtDNAs:
Circular
Small in size ~16 kb in man
5-10 copies of mtDNA / mitochondrion
~1,000 mitochondria / cell
~1% of cellular DNA
Encode:
13 proteins
large and small rRNA
tRNAs
NO INTRONS- polycistronic mRNAs
Mitochondrial genetic code has different genetic code
as compared to that in nucleus
UGA = tryptophan not STOP
AGA = STOP not arginine
AUA = methionine not isoleucine
 Mitochondrial genome encodes proteins of the
oxidative phosphorylation pathway

H+ H+ H+ H+

* subunits encoded by mtDNA

 Uncoupling proteins (UCPs)

H+ H+ H+

H+ transport into the matrix

respiration (electron transport) is uncoupled from ATP synthesis
chemical energy released as heat
siblings
Random segregation of mitochondria and mtDNA

contributed to
fertilized egg
 Threshold effect
Different tissues have different energy needs and thus,
a different levels of tolerance for mtDNA mutations

For example, if 70% of mtDNA is mutated in different

tissues is mitochondrial and cellular dysfunction
observed in all cases? Not necessarily.

Tissue Evidence of disease

Fibroblasts asymptomatic
Liver asymptomatic
Heart dysfunction
Brain dysfunction
Muscle dysfunction

Threshold sensitivity is also affected by nuclear

genetics, environment, age.
Side reaction electron transport is electron transfer to oxygen
generating oxygen radicals

O2-

O2 - O2-
 Mitochondrial electron transport chain generates
reactive oxygen species (ROS)

H++ H++
HH H+
H+
OUT Cyt C Cyt C
e - e- e- e-
e- Q e- e-

IN
e- e- +H+
H2OH O2 OH - +H
H++H
H H + 2O
H+H+- O2
HH+`+`HNAD
+ +

O2 ADP ATP
.-
O superoxide
hydroxyl .OH
radical MnSOD
NADH Krebs catalase
Cycle H2O2 H2O + O2
Fe2+ + H2O2
hydrogen
peroxide

Animated by Peter Rabinovitch

Background after Mandavilli et al, Mutation Research 509:121 (2002)
 ROS can damage DNA, proteins and lipids

OUT

IN

Krebs
Cycle

hydroxyl radicals are highly reactive

leading to damage of protein, lipids and DNA
mtDNA

Mandavilli et al, Mutation Research 509:127 (2002)

 Double agent theory of aging and disease
(Lane, J. Theor. Biol., 2003)

reactive reactive
oxygen oxygen
species species
Mitochondrial DNA and aging
 Mitochondrial DNA haplotypes associated with longevity
Masashi Tanaka and colleagues

•Accumulation of somatic mtDNA mutations is proposed to

be a major contributor to aging and degenerative diseases.
An increase in mtDNA deletions and point mutations are
detected during an individuals lifetime.

•Different mtDNA haplotypes are linked to differences in

longevity.
Study of Japanese centegenarians identified Mt5187C A
resulting in a leucine to methionine substitution the
Complex I subunit protein ND2 subunit.

The mechanism underlying increased longevity of

individuals with the Mt5187C A is not known.

Tanaka speculates that the introduced methionine may

function as an antioxidant by efficiently scavenging oxygen
radicals as proposed by Levine et al. PNAS 93:15036 (1996).
Mitochondrial DNA mutations directly linked to human disease
 Mitochondrial DNA mutations and aging

Another view:
mtDNA mutations are caused by errors in replication
not accumulated damage caused by ROS
 mtDNA REPLICATION
Synthesis of RNA primer
Mitochondrial RNA polymerase-
homology to bacteriophage RNA polymerases
single subunit
TFAM- transcription factor activator of mitochondria
TFB1M and TFB2M- mitochondrial transcription factor

Newly synthesized RNA remains hybridized to mtDNA

RNA primer is cleaved to provide 3’OH

RNase MRP (RNase mitochondrial RNA processing)

mtDNA replication requires:

POLG- mtDNA polymerase g- consists of a and b subunits
a- catalytic subunit
b- accessory subunit, primer recognition and processivity
Polymerizing activity and 3’ to 5’ exonuclease activity.
High fidelity (1 error for every 500,000 bases), proofreading capability.
Reverse transcriptase activity
mtSSB- mtDNA single stranded-DNA binding protein
Twinkle- mtDNA helicase, homology to bacteriophage T7 helicase
 Twinkle

OH
HSP and LSP- heavy and light strand promoters
mtRNA pol- mitochondrial RNA polymerase
TFAM and TFBM- transcription factors
mtSSB- mitochondrial single-strand DNA binding protein
Twinkle- DNA helicase
OH- origin of heavy strand replication
RNase MRP- RNase mitochondrial RNA processing
mtRNA pol- mitochondrial RNA polymerase
TFAM and TFBM- transcription factors
OH
 One theory of aging-
accumulation of mtDNA mutations resulting
in mitochondrial dysfunction
Transgenic mice expressing a mutant mtDNA polymerase
(POLGA- encoded by a nuclear gene)
accumulate mtDNA mutations and exhibit
premature aging and reduced lifespan
Production of homozygous knock-in mice- mtDNA mutator mice

expressing a variant of mtDNA polymerase a subunit

chromosome-encoded
catalytic subunit of mtDNA polymerase
lacking 3'-5' exonuclease activity
lacking proof-reading activity

purified recombinant polymerase has:

reduced exonuclease activity
no decrease in DNA polymerase activity
Mice express a proof-reading mutant of the mtDNA
polymerase (POLG) catalytic subunit A

Transgenic mice exhibit

- 3-5-fold increase in somatic mtDNA point mutations
- weight loss
- reduced subcutaneous fat
- alopecia- hair loss
- kyphosis- curvature of the spine
- osteoporosis
- anemia, (20% lower than wild-type)
- reduced fertility
- heart enlargement
- reduced lifespan
Demonstrates a causal link between increased somatic
mtDNA mutations and aging.
 Review (you tell me) !!!

• What genome encodes the majority of mitochondrial proteins?

• Where are these proteins synthesized?

• What are the gene products of mtDNA?

• What are the differences between mitochondrial and Mendelian

genetics?

• What are the minimal essential protein components of mtDNA

transcription and replication?

• Which reactive oxygen species are generated by mitochondria?

• Which one is the most reactive and damaging?