Mitochondrial DNA and NonMendelian Inheritance Carolyn K Suzuki

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 (n. DNA) • mitochondrial DNA (mt. DNA) 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 mt. DNA, which are synthesized in the mitochondrial matrix.

Organization of the mitochondrial genome D-loop non-coding region 16, 659 bp t. RNAs D-loop: displacement loop HSP and LSP: heavy- and light- strand promoters for transcription OH: origin of replication

Characteristics of animal mt. DNAs: Circular Small in size ~16 kb in man 5 -10 copies of mt. DNA / mitochondrion ~1, 000 mitochondria / cell ~1% of cellular DNA Encode: 13 proteins large and small r. RNA t. RNAs NO INTRONS- polycistronic m. RNAs 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 mt. DNA H+ H+

Uncoupling proteins (UCPs) 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 mt. DNA contributed to fertilized egg

Threshold effect Different tissues have different energy needs and thus, a different levels of tolerance for mt. DNA mutations For example, if 70% of mt. DNA 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 dysfunction Threshold sensitivity is also affected by nuclear genetics, environment, age.

Side reaction electron transport is electron transfer to oxygen generating oxygen radicals O 2 -

Mitochondrial electron transport chain generates reactive oxygen species (ROS) H+++ HH H H+ OUT Cyt C e- e- Q e- e- e- IN Cyt C e- e- - +H + 2 O O 2 OH H H++H H O 2 +H+ H 2 OH H+H+ + + HH+`+`HNAD ADP O 2 NADH Krebs Cycle hydroxyl radical Fe 2+ . OH ATP . O superoxide Mn. SOD + H 2 O 2 catalase H 2 O + O 2 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 mt. DNA Mandavilli et al, Mutation Research 509: 127 (2002)

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

Mitochondrial DNA and aging

Mitochondrial DNA haplotypes associated with longevity Masashi Tanaka and colleagues • Accumulation of somatic mt. DNA mutations is proposed to be a major contributor to aging and degenerative diseases. An increase in mt. DNA deletions and point mutations are detected during an individuals lifetime. • Different mt. DNA haplotypes are linked to differences in longevity.

Study of Japanese centegenarians identified Mt 5187 C resulting in a leucine to methionine substitution the Complex I subunit protein ND 2 subunit. A The mechanism underlying increased longevity of individuals with the Mt 5187 C 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: mt. DNA mutations are caused by errors in replication not accumulated damage caused by ROS

mt. DNA REPLICATION Synthesis of RNA primer Mitochondrial RNA polymerasehomology to bacteriophage RNA polymerases single subunit TFAM- transcription factor activator of mitochondria TFB 1 M and TFB 2 M- mitochondrial transcription factor Newly synthesized RNA remains hybridized to mt. DNA RNA primer is cleaved to provide 3’OH RNase MRP (RNase mitochondrial RNA processing) mt. DNA replication requires: POLG- mt. DNA 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 mt. SSB- mt. DNA single stranded-DNA binding protein Twinkle- mt. DNA helicase, homology to bacteriophage T 7 helicase

Twinkle OH HSP and LSP- heavy and light strand promoters mt. RNA pol- mitochondrial RNA polymerase TFAM and TFBM- transcription factors mt. SSB- mitochondrial single-strand DNA binding protein Twinkle- DNA helicase OH- origin of heavy strand replication

RNase MRP- RNase mitochondrial RNA processing mt. RNA pol- mitochondrial RNA polymerase TFAM and TFBM- transcription factors

OH

One theory of agingaccumulation of mt. DNA mutations resulting in mitochondrial dysfunction

Transgenic mice expressing a mutant mt. DNA polymerase (POLGA- encoded by a nuclear gene) accumulate mt. DNA mutations and exhibit premature aging and reduced lifespan

Production of homozygous knock-in mice- mt. DNA mutator mice expressing a variant of mt. DNA polymerase a subunit chromosome-encoded catalytic subunit of mt. DNA 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 mt. DNA polymerase (POLG) catalytic subunit A Transgenic mice exhibit - 3 -5 -fold increase in somatic mt. DNA 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 mt. DNA 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 mt. DNA? • What are the differences between mitochondrial and Mendelian genetics? • What are the minimal essential protein components of mt. DNA transcription and replication? • Which reactive oxygen species are generated by mitochondria? • Which one is the most reactive and damaging?