Lecture 17 & 18 DNA replication and repair Flashcards
Two strands of the DNA helix are ____.
Two strands of the DNA helix are antiparallel
the nucleotide sequence of one DNA strand is _____ to the nucleotide sequence of its partner strand
the nucleotide sequence of one DNA strand is complimentary to the nucleotide sequence of its partner strand
What are the models of DNA replication?
A) watson and crick theorised this model
B) others agreed on this model
B) others agreed on this model
An experiment was needed to show which model was correct?
[1] What is the experiment which proved that DNA is semi conservative?
1598 (meselson stahl)
- worked out a method to differentiate the new DNA from the old DNA, using a heavy isotope of Nitrogen.
- grew medium with the heavy and normal N isotope for many generations (to incorportate N into DNA)
- burst open bacterial cells and loaded contents into tubes with cesium chloride salt which created a gradient
- DNA will float or sink at the same density of the surrounding salt solution
[2] What is the experiment which proved that DNA is semi conservative? 1st Generation
- differentiate parental DNA from new DNA
- took bactieria from heavy medium and transfered to light medium (N-14)
- let it replication one round, then extracted DNA, spun in density gradient
- DNA was in an intermediate position in the tube
(now we ruled out conservative model)
[3] What is the experiment which proved that DNA is semi conservative? 2nd generation
- ruled out dispersive model
DNA acts as a ____ for its own duplication. How?
DNA acts as a template for its own duplication.
- due to complimentary nucleotide base pairing
DNA replication catalysed by DNA ____. How?
DNA replication catalysed by DNA polymerase.
- DNA polymerase catalyses the addition of nucleotides to the 3′ end
- The energy is provided by the incoming nucleotide
How does DNA replication start?
• To be used as a template:
- the double helix must be opened
- two strands separated to expose unpaired bases
What is the replication fork?
- A localised replication region moves along parental DNA helix - has a Y shape – called a replication fork
- DNA replication is bidirectional
DNA replication comes form the study of what group of organisms?
Bacteria
- the mechanisms are conserved however differ from eukaryotes
- circular chromosomes
- replication fork moves relatively fast
- Copy entire genome in around 30min
Eukaryotic chromosomes contain multiple origins of replication. What does this mean?
- reduced time for DNA replication
The DNA replication fork is ____. How?
The DNA replication fork is asymmetrical.
At each replication fork:
- one template runs in 3′ to 5′ direction
- the other template runs in the opposite 5′ to 3′direction
DNA synthesis can only occur 5′-to-3′. What does this mean for the lagging strand (going away from fork)?
- Lagging strands has the formation of Okazaki fragments made in correct 5’-3’ direction
- Leading strand – grows continuously
What are the proteins that help to open up the DNA double helix?
- For DNA synthesis to proceed: the double helix must be unzipped ahead of the replication fork
- Two types of replication proteins are needed:
- DNA helicases
- Single-strand DNA-binding (SSB) proteins
What is DNA helicase?
Uses the energy of ATP hydrolysis to propel itself forward, prying apart the double helix
What are Single-strand DNA-binding (SSB) proteins?
- the lagging strand fragemnts would natural become in an unorganised shape, making it very hard for the free nucelotides to bind
- SSB coats the strand keeping it at straight composture for free nucelotides to bind.
- bind tightly and cooperatively to ss DNA
- also called helixdestabilizing proteins
What is the accuracy of DNA replication?
- Fidelity of 5’-3’ polymerization = 1 error per 105 nucleotides
- Incorrect pairs (mispairs) can be formed
- Yet DNA polymerase only makes 1 mistake for every 107 nucleotides copied
DNA polymerase is self-correcting
Why DNA polymerase is selfcorrecting?
If DNA polymerase did nothing:
- Wrong nucleotide would be incorporated (1 in 105 )
- Frequent mutations (~30,000) would be produced
- By self-correcting mutation rate reduced to 1 in 107 (1 error for every 10 million nucleotides copied)
What are the proofreading mechanisms?
- Monitoring: DNA polymerase ‘doublechecks’ the exact base pairing
- Exonucleolytic proofreading: Error correcting reaction - DNA polymerase can correct the mismatched nucleotides
Proofreading mechanisms: how does monitoring work?
DNA polymerase undergoes conformational change more readily with correctly base-paired nucleotide.
Proofreading mechanisms: how does Exonucleolytic proofreading work?
3′-to-5’ exonuclease
- nuclease - cleaves the DNA strand*
- exo - cleaving the end*
Why does DNA replication only occur in the 5’-to-3’ direction?
only occur in the 5’-to-3’ direction allows for efficient error correction, if it was in the other direction it would block further chain elongation.
[1] How can the polymerase start replication?
The mechanism involves a different enzyme called DNA primase
- Doesn’t need base-paired end
- Uses ribonucleoside triphosphates to synthesize short RNA primers
- RNA polymerase
[2] How can the polymerase start replication, in reference to primers?
- On the leading strand : Primer is only needed at the start of replication
- On the lagging strand : New primers are needed continually
In eukaryotes: – RNA primers are about 10 nucleotides long (made at intervals of 100–200 nucleotides in eukaryotes)
Why is an RNA primer preferred to a DNA primer?
- Primase can begin new polynucleotide chains, but it does not proofread
- Primers frequently contain mistakes (1 in 105 )
- RNA primers are automatically marked as: - ‘suspect copy’ and are anyways removed and replaced by DNA
DNA ligase joins ____ .
DNA ligase joins new Okazaki fragments.
What are the 3 additional enzymes needed for lagging strand synthesis?
- nuclease – degrades the RNA primer
- repair DNA polymerase – replaces the RNA with DNA
- DNA ligase – joins the 3′ end of the new DNA fragment to the 5′ end of the previous one
What is the summary DNA replication?
The proteins at a replication fork cooperate to form a replication machine
How is the lagging strand shaped?
The lagging strand is actually folded
- allows for the lagging strand to have access to the complex of proteins when replication occurs
What is a sliding ring?
holds a moving DNA polymerase onto the DNA
An accessory protein (PCNA in eukaryotes):
- Functions as a regulated sliding clamp
- Forms a large ring around DNA double helix
- Keeps the polymerase firmly on the DNA
- Increases processivity of the DNA polymerase
What is a clamp holder?
- Assembly of the clamp around DNA requires ATP hydrolysis by the clamp loader
- Leading strand - loading needs to occur once
- Lagging strand – the clamp is removed and reattached at the start of each new Okazaki fragment
What is the “winding problem” refering to?
The “winding problem” arises during DNA replication
If the tension is not relieved the DNA will become overwound and supercoiled.
[1] What is function of DNA topoisomerases?
to relieve the tension and prevent supercoiling
.
[2] What is DNA topoisomerase I?
produces a transient singlestrand break
[3] What is DNA topoisomerase II?
makes a transient double-strand break - Prevents tangling
What is an end-replication problem?
- DNA replication proceeds only in the 5′-to-3′ direction.
- Lagging strand is synthesized in discontinuous fragments.
• A special problem arises: as the replication fork reaches the end of a chromosome
lagging strand would get shorter with each round of DNA replication
What is the end-replication problem in bacteria?
This is not a problem beause DNA is circular and there are no ends
What is Telomerase?
- Eukaryotes “end-replication” problem:*
- have special repetitive nucleotide sequences at ends of chromosomes (GGGTTA in humans)
- incorporated into structures called telomeres
- attract an enzyme called telomerase
- elongates telomere DNA in the 5′-to-3′ direction
How does Telomerase function?
Telomerase replicates the ends of chromosomes
Telomerase is a _____.
Telomerase is a reverse transcriptase.
making DNA from RNA template
What is present at the end of a mammalian chromosome?
A t-loop at the end of mammalian chromosomes to product ends of chromosomes → Cells can distinguish between ends of chromosomes and double-strand DNA breaks
prevents single transded dna from degrading, it is folded on itself and tucked inside
What is the importance of DNA repair?
- Genetic stability requires accurate replication, but also repair mechanisms
- Most spontaneous changes in DNA are immediately corrected by a set of processes called DNA repair.
- Importance of DNA repair is demonstrated by:
– Large investment in DNA repair enzymes
– Increased mutation rate following inactivation of a DNA repair gene
A mismatch repair system removes ____.
A mismatch repair system removes replication errors.
Mismatch repair corrects 99% of replication errors
How does them mismatch repair system work?
detects the potential for distortion in the DNA helix
• Must be able to distinguish between newly synthesized strand and original DNA strand
- The two proteins involved in mismatch repair (MutS and MutL) are present in both bacteria and eukaryotic cells
- e.g. MSH2 (MutS) mutations predispose to colon cancer*
What are spontaneous DNA changes?
Sources of DNA damage:
- Endogenous e.g. reactive metabolites (ROS, H20), replication errors.
- Exogenous e.g. UV radiation, x-rays, mutagenic chemicals
Without DNA repair, what would happen to spontaneous DNA damage?
What are the two examples of the most frequent cause of damage?
spontaneous DNA damage would rapidly change DNA sequences
- Depurination
- Deamination
- Thymine dimers (UV radiation)
What is Depurination?
Depurination removes a purine base from a nucleotide
Depurination - What happens when there is a baseless DNA region?
What is deamination?
results in spontaneous loss of an amine group e.g. converts a cytosine base to a uracil
Most common example is with cytosine
Deamination - what happened if this error was not corrected?
Deamination - what happenes if the error goes undetected by the body?
Mutation “hotspots”
can affect how and when the gene is expressed
What are Thymine dimers?
relates to UV radiation
Thymine dimers covalent linkage between adjacent pyrimidine bases.
two nucleotides fuse together
What are the 2 pathways to remove DNA damage?
- Base excision repair (BER)
- Nucleotide excision repair (NER)
in both:
- the damage is excised (nuclease)
- the original DNA sequence is restored (repair DNA polymerase)
- the double helix is sealed (DNA ligase)
Base excision repair (BER) - How is the damage recognized?
- recognize specific, non-bulky lesions in DNA.
- damaged bases removed by specific glycosylases.
- creates AP sites, with a baseless sugar. e.g. U is restored to a C
Nucleotide excision repair (NER) - How is the damage recognized?
- repairs bulky, helixdistorting DNA damage
- bacteria - 12 nucleotide gap
- human - ~30 nucleotide gap
- Global vs transcriptioncoupled
e. g. individuals with xeroderma pigmentosum, Cockayne syndrome
What happens when a double strand breaks and what are 2 mechanisms for repair?
- Double-strand breaks can lead to fragmentation of chromosomes and loss of genes
- Especially difficult to repair as each chromosome contains unique information
- 2 distinct mechanisms have evolved to deal with this type of damage:
- non-homologous end-joining
- homologous recombination
How do the mechanisms if a double strand breaks work?
What is Homologous recombination?
- Genetic exchange takes place between a pair of homologous DNA sequences
- Can only occur after DNA has been replicated and before the cell divides.
- e.g. breast cancer caused by mutations in BRCA1 or BRCA2
Examples of severe consequences due to failure to repair DNA damage?
Mutations in germ cells can lead to inherited diseases
- Sickle-cell anaemia A permanent change in a single nucleotide in the haemoglobin gene –> Haemoglobin with an incorrect sequence of amino acids
- Palmoplantar keratoderma (skin damage)
- Cancer mutations in somatic cells (The chance that a cell will become cancerous greatly increases with age)
