Flashcards in DNA Replication 2 - Eukaryotes Deck (32):
Why is it important to understand the aspects of mitochondrial DNA?
Mitochondrial DNA is associated with disease states related to metabolism and energy production. Recent studies indicate that mitochondrial dysfunction may be more prevalent than previously thought (1/4300 live births lifetime risk).
What are the 3 unique aspects of mitochondrial DNA replication?
1. Each mitochondrion contains 2 to 10 copies of the approximately 17,000 base pairs in a circular genome that are not evenly distributed during cellular division.
2. Many of the proteins required for mitochondrial function are nuclear encoded, such as the enzyme responsible for mitochondrial DNA replication (DNA polymerase gamma).
What is Heteroplasmy in relation to mitochondrial DNA?
Heteroplasmy occurs when one cell contains multiple types of mitochondrial DNA with varying levels of each type from one cell to the next. Thus, unlike nuclear DNA (constant from one cell to the next), mitochondrial DNA can vary from one cell to the next, causing a complicated disease pattern when deleterious mutations are present.
There is a link between Heteroplasmy and mutations with strong dysfunction. What are some of the diseases and why are they rare?
1.Diseases include myoclonic ataxia and epilepsy with ragged red fibres (MERRF), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), and Kearn-Sayre syndrome.
2. Most are de novo mutations and the afflicted individual does not live beyond childhood so they do not pass on the mutation.
Where do mutations that cause weak or moderate dysfunction tend to occur?
What is an example of each?
1. Mutations that cause weak or moderate dysfunction tend to occur in a Homoplastic state when exerting an effect on disease. Weak dysfunction alleles tend to only be linked with disease and may exert their effects in combination with environmental factors like smoking.
2. Weak = Parkinson's
Moderate = Leber hereditary optic neuropathy (LHON)
What is mitochondrial DNA depletion syndrome?
Mitochondrial DNA depletion syndrome is progressive disease affecting children and those treated for HIV (AZT drug is nucleotide analog that targets viral replication but may also interact with DNA polymerase gamma).
Metabolic defects in replication of mitochondrial DNA result in deletion of this DNA and associated pathology.
What is the pathology of mitochondrial DNA depletion syndrome?
1. 13 members of the ETC are encoded in mitochondrial DNA. Their loss inhibits oxidative phosphorylation.
2. Prominent hepatic abnormalities may be explained by the dramatic postnatal developmental changes and mitochondrial adaptation that occurs in this organ in the first few months and years of life.
3. Postnatal adaptive changes in skeletal muscle mitochondria occur only later.
What do the non-hepatic (more encephalomyopathic) forms of mitochondrial DNA depletion reflect?
Encephalomyopathic forms may reflect early disturbances in the myogenic program that result in either destabilization of the muscle cell membrane or physical muscle cell breakdown with measurable increases in creatine kinase (CK) from muscle.
Muscle, liver, and brain all undergo encounters with the postnatal environment.
What is the significance of the high ratios of NADH to NAD+ found in the severe deficiencies of complexes I, III, and IV in a patient with mitochondrial DNA depletion?
This should decrease mitochondrial beta oxidation and provide a mechanism for impaired fatty acid oxidation.
What is the "red flag" clinical presentation of mitochondrial disorders?
The "red flag" is multi-organ disturbance with the highly energy-demanding organs (brain, liver, skeletal muscle) involved.
What diseases have been linked to mitochondrial DNA depletion?
Alpers syndrome, progressive external opthalmoplegia, and ataxia-neuropathy syndrome are linked to mitochondrial DNA depletion. (Aging is also associated with mitochondrial DNA damage.)
The domains of the mitochondrial targeting sequence are associated with what functions?
1. Exonuclease domain = proofreading
2. Linker region = lower protein activity
3. Polymerase domain = elongation
(See schematic on page 21 of Notes)
What is the major difference between eukaryotic and prokaryotic DNA replication in regards to time and space?
Eukaryotic DNA synthesis occurs from numerous origins of replication, spaced about every 30,000 to 300,000 base pairs and the replication forks proceed at 1/20th the pace of prokaryotic replication forks.
What is the major difference between eukaryotic and prokaryotic polymerases?
Eukaryotic polymerases are denoted with Greek letters rather than Roman numerals.
What is the function of eukaryotic DNA polymerase alpha?
DNA polymerase alpha contains Primase (lays down primer) and initiates DNA synthesis. (Other enzymes are then recruited to continue synthesis.)
What is the function of eukaryotic DNA polymerase beta?
DNA polymerase beta is involved in DNA repair.
What is the function of eukaryotic DNA polymerase gamma?
DNA polymerase gamma replicates mitochondrial DNA and has 3' --> 5' proofreading activity.
What is the function of eukaryotic DNA polymerase delta?
DNA polymerase delta synthesizes the lagging strand and has 3' --> 5' proofreading activity.
What is the function of eukaryotic DNA polymerase epsilon?
DNA polymerase epsilon substitutes for DNA polymerase delta in some situations such as DNA repair and leading strand elongating. It also has 3' --> 5' proofreading activity.
Which eukaryotic DNA polymerases are the workhorses of DNA replication?
The workhorses of DNA replication are those eukaryotic DNA polymerases that have proofreading activity.
These include gamma, delta, and epsilon.
How is nuclear DNA replication in eukaryotes linked to cellular division in the cell cycle?
DNA replication is occurring in S phase (Synthesis phase). This requires a huge amount of energy.
What problems are caused by the linearity of eukaryotic nuclear chromosomes?
1. The ends must be protected from nuclease degradation in order to maintain integrity of the genome.
2. The ends must be distinguished from double stranded DNA breaks in order to prevent chromosomal fusion events.
3. Full replication of the ends is important in cellular senescence (aging) and cellular crisis (cancer).
What addresses the problems caused by the linearity of eukaryotic nuclear chromosomes?
Telomeres address the problems caused by the linearity.
What is cellular senescence?
Cellular senescence is aging of the cell. Telomeres shorten with each cellular division, thereby acting as a mitotic clock in that the cell can only divide so many times until the telomeres become so short that it no longer divides and can enter cellular crisis.
What is cellular crisis?
Cellular crisis occurs when the cell has divided so many times that the telomeres are too short for it it divide any longer. The end result is cell death and cytogenetic abnormalities that cause the chromosomal instability associated with cancer.
What is the structure of telomeres?
The telomere structure contains a buffer region of 100,000 to 300,000 bp telomere-associated repeats followed by a 3,000 to 20,000 bp region of tandem TTAGGG repeats then a single stranded region of several hundred base pairs folded upon itself and bound by proteins.
Why does the telomere shorten every time the nuclear genome is replicated?
The telomere is shortened due to the necessity of an RNA primer for DNA replication.
What is Telomerase?
Telomerase is a special DNA polymerase that contains an RNA molecule and functions as the template for the telomeric repeat.
What kind of polymerase is Telomerase?
Telomerase is a Reverse Transcriptase, because it depends on RNA to transcribe DNA.
What are the locations and functions of Telomerase?
1. Telomerase is present in stem cells to prevent shortening of the ends of the chromosomes.
2. Telomerase is activated in cancer cells to immortalize them.
What is the mechanism of action of Telomerase?
1. Telomerase uses its Reverse Transcriptase activity to extend the 3' end of ssDNA.
2. Primase synthesizes an RNA primer.
3. The 3' end of the RNA serves as an acceptor for polymerase to extend the RNA primer.
4. The RNA primer is removed, resulting in a net preservation or extension of the telomeric 5 bp repeat.
(See page 24 of Notes)