Mod 2- DNA Replication Flashcards
What are the different types of DNA replication?
Semiconservative
Conservative
Dispersive replication
What does semiconservative replication entail?
The two DNA strands separate and DNA is copied using the parent strand as a template
Ends up with 2 identical strands same as parent DNA- each daughter strand has half the information from the parent.
Why is DNA replication essential?
To pass on equal amounts of DNA when cells divide
Defects can lead to diseases like cancer.
What role do topoisomerases play in DNA replication?
They solve the problem of DNA double helix unwinding during replication
Allow for efficient and accurate replication.
What was the initial belief about semiconservative replication?
It was thought to be impossible due to DNA being plectonemic (Strands can’t be separated without unwinding the double-helix structure)
What did the Meselson-Stahl experiment demonstrate?
That DNA is replicated by the semiconservative process in living cells
Conducted in 1959 using heavy nitrogen.
What is the purpose of density gradient centrifugation in the Meselson-Stahl experiment?
To distinguish between 15N-DNA and 14N-DNA by buoyant density
Cesium chloride (CsCl) was used.
What are DNA supercoils, and why must they be managed?
Produced if strands are separated; they need to be removed or prevented
Managed by DNA topoisomerases.
What is the function of Type I DNA topoisomerase?
Separates the strands without unwinding the DNA double helix
What is the G segment in Type II topoisomerase action?
The DNA strand that gets completely cut to allow another strand to pass through.
What is the T segment in Type II topoisomerase action?
The DNA strand that gets passed through
Essential for managing DNA replication.
Fill in the blank: DNA replication is essential for _______.
[reproduction and continued life]
True or False: Conservative replication was the first hypothesis believed to be true about DNA replication.
True
Scientists initially thought semiconservative replication was impossible.
What is the method of DNA replication that ensures copies are made exactly without errors?
semiconservative
This method allows each new DNA molecule to contain one original strand and one newly synthesized strand.
What are the cellular enzymes that synthesize DNA strands from nucleotides called?
DNA polymerases
DNA polymerases play a crucial role in DNA replication and repair.
What is the direction of DNA synthesis during replication?
5’ to 3’
DNA synthesis always occurs in this direction, requiring a primer to initiate.
What is the role of a primer in DNA synthesis?
To initiate synthesis of the new strand
Without a primer, DNA synthesis cannot occur.
What enzyme is responsible for making the RNA primer in bacteria?
primase
The primer is typically 4-15 nucleotides in length.
What is the term for the short sections of DNA synthesized on the lagging strand?
Okazaki fragments
These fragments are later joined together to complete the lagging strand.
What type of activity allows DNA polymerases to correct errors during DNA synthesis?
exonuclease activity
This includes 3’ to 5’ proofreading, where the polymerase removes incorrectly inserted nucleotides.
What protects the separated single strands of DNA during replication?
single-strand binding proteins (SSBs)
These proteins prevent the strands from reattaching or being degraded by nucleases.
In eukaryotes, which DNA polymerase first extends the RNA primer?
DNA pol alpha
DNA pol alpha adds about 20 nucleotides before DNA pol delta synthesizes the rest.
True or False: DNA replication can occur in both directions at the replication fork.
False
DNA replication can only occur in one direction, but the two strands are oriented oppositely.
What happens to the RNA primers after Okazaki fragments are synthesized?
They are removed and the fragments are joined together
This process is essential for completing the lagging strand synthesis.
What is the purpose of DNA topoisomerases during DNA replication?
To relieve the stress of DNA double-helix unwinding
They nick or cut the DNA to facilitate this process.
What is the first step in synthesizing a new strand of DNA at the replication fork?
The creation of a primer
This primer is necessary for initiating DNA synthesis.
What is the enzyme involved in joining Okazaki fragments in eukaryotes?
FEN1
FEN1 is an endonuclease that plays a critical role in this process.
What are the two types of strands during DNA replication?
Leading strand and lagging strand
What is the end replication problem?
DNA molecules gradually getting shorter with each round of replication
What is the role of telomerase?
Prevents the ends of chromosomes from being shortened
It extends the parent DNA by adding a sequence (TTAGGG) several times
When does DNA replication take place?
During the S phase of the cell cycle
What is the leading strand?
The strand that is copied by continuous DNA synthesis
Copying of this strand requires a primer
What is the primer made of in bacteria?
RNA
What enzyme synthesizes the primer in bacteria?
Primase
(On leading strand)
How long is the RNA primer in bacteria?
4-15 nucleotides in length
Which DNA polymerase extends the RNA primer in eukaryotes?
DNA pol alpha
Adds about 20 nucleotides
Has no exonuclease activity
What is the function of DNA pol delta in eukaryotes?
Makes the rest of the new strand after DNA pol alpha
Has 3’—>5’ exonuclease proofreading activity
How is the lagging strand copied?
From a primer placed at the replication fork
In what direction is DNA synthesis always carried out?
5’ → 3’ direction
What are the sections of the lagging strand called?
Okazaki fragments
What must be done to complete lagging strand synthesis?
Join Okazaki fragments and remove RNA primers
What enzyme is involved in joining Okazaki fragments in eukaryotes?
FEN1 (an endonuclease)
What is the sequence added by telomerase?
TTAGGG
This is unique to each organism but is this sequence in humans
What type of complex is telomerase?
RNA/protein complex
Why is telomerase particularly important in Tetrahymena thermophila?
It has a large number of chromosomes (40,000)
What is senescence in cell culture?
Cells can divide about 50 times before dying
What happens to chromosome ends in senescent cells?
They become shortened
During which phase of the cell cycle does DNA replication occur?
S phase (synthesis phase)
Lasts about an hour/ couple of hours
What can be visualized to detect sites of DNA replication?
Modified nucleotides incorporated into DNA and detected with fluorescently labelled antibodies to visualise sites of DNA replication
What are the two types of DNA that are replicated during S phase?
- Euchromatin = open, active DNA where genes get transcribed and are used
- Heterochromatin = condensed, inactive DNA where genes aren’t expressed or being used
How does Type I DNA topoisomerase work?
- Nicks the DNA once
- Cuts it at one strand
- Enzyme pulls strand through
- Joins again by ligase
- So less helical (one fewer ‘twist’)
What is the mechanism for Type II topoisomerase?
Major enzyme used
- Double strand is cut
- Pass second segment of the DNA molecule through the gap (another double strand)
- Rejoin the two cut strands
How are new strands of DNA synthesised at the replication fork?
By template-dependent DNA synthesis (ensures identical copy of DNA)
The template is DNA so this is DNA-dependent DNA synthesis
What is exonuclease activity?
Where DNA polymerase degrades DNA
Acts on ends
What are the 2 possible types of exonuclease activity?
3’—>5’ exonuclease activity
- going in opposite direction to synthesis
- the polymerase can remove nucleotides it has just inserted
- this is called proofreading = allows errors to be corrected (removes any incorrect bases that have been inserted = important to accuracy)
5’—>3’ exonuclease activity
- the polymerase can remove DNA already attached to the template
Does DNA pol Alpha have exonuclease activity?
No
It’s function is priming during replication
Does DNA pol Delta have exonuclease activity?
Yes
ONLY 3’—>5’ exonuclease activity (proofreading)
This is the main replicative enzyme
What happens at the replication fork?
Replication occurs
Separated single strands must be protected, if not
- they may just reattach to one another
- they might be attacked by nucleases
The strands are protected by single-strand binding proteins (SSBs)
What happens once the primer is made in prokaryotes?
DNA pol III makes the new strand
DNA pol III has a 3’—>5’ exonuclease proofreading activity
What happens when the replication fork moves?
The leading strand is extended by more DNA synthesis (continued synthesis)
The lagging strand has to be made in sections = requires multiple primers (ensures 5’ to 3’ direction)
Describe the roles and structure of bacterial DNA polymerases
DNA pol I
- has 1 subunit
- Has 3’ to 5’ AND 5’ to 3’ exonuclease activity
- Function = DNA repair and replication
DNA pol III
- has at least 10 subunits
- has 3’ to 5’ exonuclease activity ONLY
- Function = main replicative enzyme
Describe the roles and structure of Eukaryotic DNA polymerases
DNA pol Alpha
- has 4 subunits
- NO exonuclease activity
- Function = priming during replication
DNA pol Delta
- has 2 or 3 subunits
- has 3’ to 5’ exonuclease activity ONLY
- Function = main replicative enzyme
How are Okazaki fragments in eukaryotes joined?
- DNA pol delta and helicase push aside the primer
- forms a ‘flap’
- FEN1 (flap endonuclease) cuts in the middle of the molecule at the branch point
- leaving a missing phosphodiester bond
- DNA ligase links the two DNA fragments
How are Okazaki fragments in bacteria joined?
- DNA pol III stops when it reaches the RNA primer (as no exonuclease activity)
- DNA pol I (has 5’—>3’ exonuclease activity) continues synthesis until it gets rid of all the RNA
- DNA ligase links the two DNA fragments
Why can the final Okazaki fragment not be made in eukaryotes?
Because the priming site would be after the end of the parent molecule which would lead to DNA molecules gradually getting shorter
How does telomerase act as an extension at the end of a human chromosome?
Telomerase is an RNA/protein complex
It acts as a template
A few thousand telomerase repeats at the end of human chromosomes TTAGGG acts as a big buffer
(RNA sequence is AAUCCC)
Do all cells have telomerase?
No, only stem cells have telomerase
(Turns off telomerase activity once cells differentiate)
What do the patterns of DNA replication observed during S phase show?
Patterns reflect the different types of DNA being replicated at different times of the cell cycle
