17. THE MITOCHONDRIAL GENOME

Question 1

What are the 7 features of the mitochondrial genome?

Answer 1

1. MtDNA is a double stranded & circular molecule
2. mtDNA consists of a heavy chain & a light chain
3. It’s mult-copy genome consisting of 37 genes: 13 = OXPHOS, 22 = tRNA, 2 = rRNA
4. mtDNA has no introns
5. It has a d-loop structure which is a non-coding region where DNA transcription & replication is initiated
6. mtDNA is maternally inherited so there’s no recombination
7. mtDNA encodes proteins of oxidative phsophorylation

Question 2

Where does mtDNA replication & transcription start?

Answer 2

• The non-coding region of mtDNA is responsible for replication & transcription. The D-loop is located in the non-coding region
• mtDNA REPLICATION - replication of the heavy strand occurs at the OH (Origin of the heavy chain), replication of the light chain occurs at the OL (Origin of the light chain)
• mtDNA TRANSCRIPTION - starts at the heavy chain promoter & the light chain promoter

Question 3

How is mtDNA packaged?

Answer 3

• mtDNA is packaged as NUCLEOIDS
• There are 1-2 copies of mtDNA in the nucleoid
• It also consists of transcription factor A (TFAM) , which acts as a histone protein

Question 4

What are the mtDNA exceptions to the genetic code?

Answer 4

• AUG & AUA code for methionine in mtDNA but AUA codes for isoleucine in the genetic code
• UGA codes for tryptophan in mtDNA but it’s a stop codon in the genetic code
• AGA & AGG are stop codons in mtDNA but arginine is the stop codon in genetic code

Question 5

What are mtDNA haplogroups?

Answer 5

• mtDNA haplogroups refers to different mitochondrial variants among the populations

Question 6

How many OXPHOS proteins does mtDNA code for?

Answer 6

• Mitochondria requires both nuclear & mitochondrial DNA
• 13 OXPHOS proteins are coded by mitochondrial DNA
• There are over 1000 OXPHOS proteins but the rest of them are coded by nuclear DNA
• The mitochondrial DNA needs to be replicated, transcribed & translated

Question 7

What is the mitochondrial DNA polymerase involved in replication & what does it consist of?

Answer 7

• The mitochondrial DNA polymerase is known POLYMERASE GAMMA (PolY)
• It consists of:
  1. Heterodimer protein
  2. One catalytic sub unit - PolY A
  2. Two accessory sub-units - PolY B -

Question 8

What does Polymerase gamma A do?

Answer 8

• Polymerase gamma A is a catalytic sub-unit if polymerase gamma
• It contains a 3’5 exonuclease which proof reads newly synthesised DNA

Question 9

What does Polymerase gamma B do?

Answer 9

• Polymerase gamma B is an accessory subunit

- It enhances interactions with the DNA template to increase Polymerase gamma A

Question 10

What is the mitochondrial DNA helicase & what does it do?

Answer 10

• TWINKLE is the mitochondrial DNA helicase
• It is a hexamer made up of 6 subunits
• It unwinds double stranded mtDNA template to allow Polymerase gamma to carry out it’s replication

Question 11

What 3 things does mitochondrial single stranded binding protein do?

Answer 11

• Mitochondrial single stranded binding protein binds to single stranded DNA which:
  1. Protects against nucleases
  2. Prevents secondary structure formation
  3. Enhances mitochondrial DNA synthesis by stimulating TWINKLE

Question 12

What are the 4 steps of the strand displacement model of mitochondrial DNA replication?

Answer 12

• Strand displacement model:
  1. Replication of heavy strand at OH
  2. Replication of light strand at OL
  3. Replication of both strands completed
  4. Segregation of daughter molecules

Question 13

Describe the replication of the heavy strand

Answer 13

1. At OH, the parental strand is displaced & is coated with mtSSB (mitochondrial single stranded binding protein)
2. This allows TWINKLE to unwind the double stranded DNA
3. Mitochondrial RNA polymerase (POLRMT) produces an RNA primer using the light chain as a template
4. Polymerase Gamma uses the RNA primer to replicate the DNA at OH

Question 14

Describe the replication of the light strand

Answer 14

1. As replication of the heavy strand proceeds, the light strand is reached
2. A stem loop structure is formed which prevents mitochondrial single stranded proteins (mtSSB) from binding
3. This gives access to the mitochondrial RNA polymerase (POLRMT) which synthesises an RNA primer using the light strand as a template
4. Polymerase gamma uses the RNA primer to replicate light strand at OL

Question 15

What enzyme is needed for the segregation of daughter molecules produced by mtDNA replication?

Answer 15

• After DNA replication, the daughter molecules are separated or segregated by the topoisomerase protein TOP 3A
• In the absence of Top 3a protein, the molecules are catenated meaning they’re joined together

Question 16

What are the features of mitochondrial diseases & give examples?

Answer 16

• Mitochondrial diseases are rare, monogenic diseases
• Oxidative phosphorylative diseases are the most common
• Leigh’s syndrome

Question 17

What are 5 ways in which mitochondrial diseases can be diagnosed?

Answer 17

1. Clinical signs
2. Neuroimaging
3. Histochemical analyses
4. DNA analysis
5. Enzymatic assay of OXPHOS complexes

Question 18

What are the three biochemical measurements for mitochondrial diseases?

Answer 18

1. Blood/CSF lactic acid raised due to pyruvate build up metabolised to lactic acid as a result of OXPHOS diseases
2. Lactic acid/Pyruvate ratio
3. AA & Organic acids

Question 19

How can muscle biopsies be used to show mitochondrial diseases?

Answer 19

• Muscle biopsies can be taken
• Histochemical of the muscle mitochondria can show mitochondrial disease
• GOMORI TRICHOME - Used to visualise mitochondria. Ragged red fibers indicate an accumulation of mitochondria

Question 20

What is homoplasmy & heteroplasmy?

Answer 20

• Homoplasmy = refers to the presence of one type of mtDNA
• Heteroplasmy = refers to the presence of more than one type of mtDNA
• Can be pathogenic or healthy variant

Question 21

How do heteroplasmy levels determine whether an individual develops a mitochondrial disease?

Answer 21

• Heteroplasmy levels determines whether an individual develops a disease or not
• The quantity of mtDNA variants in a cell determines the likelihood of developing the gene
• If 80% of the cell DNA is pathogenic mtDNA variants, the disease manifests but it also depends on the type of variant
• Heteroplasmy levels also affect penetrance & the severity of the disease

Question 22

How can mitochondrial diseases be inherited?

Answer 22

• Mitochondrial diseases caused by mtDNA mutations are inherited maternally from the mother
• The disease can only be passed on by females. Males who are affected cannot pass it on
• However, in some cases an individual will have an mtDNA disease without having an affected parent due to heteroplasmy
• Heteroplasmic mutations are inherited by chance & are passed on in variable amounts
• Homoplasmic mutations are passed on to all offspring. Females affected will pass it on to all their children e.g LHON
• Mitochondrial diseases can also be caused by nuclear mutations, de novo mutations

Question 23

What do mutations in mtDNA replication machinery lead to?

Answer 23

• Can lead to mtDNA deletions or duplications

- Likely to occur in post-mitotic tissues such as the brain, heart, liver & muscle

Question 24

What do mutations in TWINKLE cause?

Answer 24

Can lead to mtDNA deletions in brain, muscle 7 heart

- Can lead to late-onset mitochondrial myopathy or progressive external opthamophlegia (drooping of the eyelids)

Question 25

Give 4 ways that mtDNA mutation transmission can be prevented?

Answer 25

1. Oocyte donation
2. Pre-natal diagnosis
3. Pre-implantation diagnosis (IVF)
4. Mitochondrial replacement therapy