1. Mitochondrial DNA and Non-Mendelian Inheritance Carolyn K. Suzuki, Ph.D. Dept of Biochemistry

2. Essential functions of mitochondria Biosynthesis of amino acids nucleotides steroid hormones heme ATP synthesis Oxidation of fatty acids Apoptotic cell death

3. Mitochondrial proteins are encoded by 2 separate genomes and translated by 2 different protein synthesis machineries

4. 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.

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

6. 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

7. Mitochondrial genome encodes proteins of the oxidative phosphorylation pathway * subunits encoded by mtDNA H+H+ H+H+ H+H+ H+H+

8. Uncoupling proteins (UCPs) H + transport into the matrix respiration (electron transport) is uncoupled from ATP synthesis chemical energy released as heat H+H+ H+H+ H+H+

10. siblings

11. Random segregation of mitochondria and mtDNA contributed to fertilized egg

13. Different tissues have different energy needs and thus, a different levels of tolerance for mtDNA mutations Threshold effect For example, if 70% of mtDNA is mutated in different tissues is mitochondrial and cellular dysfunction observed in all cases? Not necessarily. TissueEvidence of disease Fibroblastsasymptomatic Liver asymptomatic Heartdysfunction Braindysfunction Muscledysfunction Threshold sensitivity is also affected by nuclear genetics, environment, age.

14. Side reaction electron transport is electron transfer to oxygen generating oxygen radicals O2-O2- O2-O2- O2-O2-

15. Krebs Cycle e-e- e-e- e-e- e-e- H+H+H+H+ H+H+ H+H+H+H+H+H+ H+H+ H+H+ H+H+H+H+ H+H+ H+H+H+H+H+H+ H+H+ H+H+ Cyt C e-e- e-e- H+H+H+H+H+H+H+H+ H+H+H+H+H+H+H+H+ O2O2 H+H+H+H+H+H+H+H+ H+H+H+H+H+H+H+H+ H2OH2O ADPATP Cyt C e-e- O2O2 Q H +` H + NADH NAD + H+H+H+H+ H+H+H+H+ H + H + H + H + H + Cyt C H +` H +` e-e- O2O2 e-e- e-e- IN OUT Animated by Peter Rabinovitch Background after Mandavilli et al, Mutation Research 509:121 (2002) Mitochondrial electron transport chain generates reactive oxygen species (ROS) 2OH - OH - H2O2H2O2 H2O2H2O2 Fe 2+ + O-O-. superoxide MnSOD O-O-. H2O2H2O2 hydrogen peroxide. OH hydroxyl radical H2OH2O O2O2 + catalase

16. IN OUT Mandavilli et al, Mutation Research 509:127 (2002) ROS can damage DNA, proteins and lipids mtDNA Krebs Cycle hydroxyl radicals are highly reactive leading to damage of protein, lipids and DNA

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

18. Mitochondrial DNA and aging

19. 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.

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

21. Mitochondrial DNA mutations directly linked to human disease

22. Mitochondrial DNA mutations and aging Another view: mtDNA mutations are caused by errors in replication not accumulated damage caused by ROS

23. 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  - consists of a and b subunits  - catalytic subunit  - 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 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 mtDNA REPLICATION

24. 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 O H - origin of heavy strand replication OHOH Twinkle

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

26. OHOH

27. One theory of aging- accumulation of mtDNA mutations resulting in mitochondrial dysfunction

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

29. 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

30. 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.

31. 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?