Download Lecture 35

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+
* subunits encoded by mtDNA
H+
H+
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
Fibroblasts
Liver
Heart
Brain
Muscle
Evidence of disease
asymptomatic
asymptomatic
dysfunction
dysfunction
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
e - e-
Q e- e-
e-
IN
Cyt C
e- e-
e- e-
- +H
+ 2O
O2 OH
H
H++H
H
O2
+H+
H2OH
H+H+-
+
+
HH+`+`HNAD
ADP
O2
NADH
Krebs
Cycle
hydroxyl
radical
.OH
ATP
.O superoxide
MnSOD
catalase
Fe2+
+ H2O2
H2O2
H2O + O2
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
oxygen
species
reactive
oxygen
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
resulting in a leucine to methionine substitution the
Complex I subunit protein ND2 subunit.
A
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 polymerasehomology 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 agingaccumulation 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?