Exam 2-3 Flashcards
Problem: No single stranded template.
What is the solution? Is ATP involved?
Solution: Helicase.
ATP is involved.
In what direction does a helicase unzip DNA in eukaryotes? What about prokaryotes?
Eukaryotes : 3’ -> 5’ strand (leading strand)
Bacteria : 5’ -> 3’ strand (lagging strand)
How does a helicase work?
Helicases need a ton of ATP to open up DNA.
When ATP is bound to the helicase, the loops on the helicase interact with the phosphodiester bond of DNA via ATP hydrolysis.
It changes the shape of DNA, pulling it down and allowing helicase to unzip the DNA strand and disrupting the H-bonding between bases.
Problem: The single stranded template is unstable.
What is the solution? Is ATP involved?
Solution:
SSB: Single-stranded binding protein(bacteria)
RPA: Replication Protein A (eukaryotes)
What is the role of SSB in replication?
It binds to single stranded DNA, protecting it from annealing and folding into unwanted forms.
How does SSB perform their role?
They bind to the backbone of the phosphodiester bonds of DNA. This will make the bases more available for DNA polymerase.
Problem: No primer.
What is the solution? Is ATP involved?
Solution: Primase
ATP: (+)
Explain the role of the primase.
Primase is an enzyme that is going to really begin polymerization.
It is an RNA polymerase that is going to add ribonucleotides to the first sets of nucleotides on a strand. The primer that is set down acts as a starting point of DNA.
The polymerase starts replication at the 3’-end of the primer, and copies the opposite strand.
Problem: No 3’-> 5’ polymerase.
What is the solution? Is ATP involved?
Solution: Replication Fork
ATP is not involved.
DNAP III dimers couple leading and lagging strands.
Beta-clamps go over primer units and helps DNA polymerase stay on the DNA strand.
Multiple clamps are used for the lagging strand since it is synthesized in fragments.
Problem: Too slow and distributive.
What is the solution? Is ATP involved?
Solution: SSB and Sliding Clamp.
ATP is involved.
How do SSB and sliding clamps help solve the problem of DNA synthesis being too slow or distributive?
SSB help to keep DNA linear, preventing it from folding and annealing on itself.
The sliding clamp ensures that DNA Polymerase III stays on the DNA strand for replication.
How does the process of getting the sliding clamp onto the DNA molecule?
When a DNA clamp loader binds to ATP, It opens up its ring structure.
The clamp loader finds a sliding clamp/beta clamp and binds to that protein, opening its ring structure as well.
The clamp loader than takes the complex to a DNA molecule where the hydrolysis of ATP forces the clamp to close, sealing the ring structure of the sliding clamp over the DNA molecule.
The sliding clamp/beta clamp has subunits that attracts DNA polymerase III, keeping the polymerase on DNA until replication is finished.
Problem: Lagging Strand contains RNA.
What is the solution? Is ATP involved?
Solution: DNA Polymerase I -> 5’ -> 3’ Exonuclease activity and RNase H.
Does not require ATP.
What is the role of 5’ -> 3’ Exonuclease activity in DNA polyermase I and RNase H?
They are responsible for the removal of RNA primers for the addition of DNA nucleotides.
RNase H removes the RNA primer created by Primase. Polymerase I fills in the necessary nucleotides between the Okazaki fragments in the 5’ -> 3’ direction.
Problem: Lagging strand is nicked.
What is is the solution? Is ATP used?
Solution: DNA Ligase
ATP is used.
What molecule is needed to repair a nick via DNA Ligase?
ATP (Eukaryotes and viruses)
NAD+ (Bacteria)
Explain the 3-Step mechanism of DNA Ligase.
- Adenylation of DNA liagase.
- Addition of AMP or NAD+ onto DNA ligase to “activate it”. - Activation of 5’ Phosphate in the nick, with the addition of AMP/NAD+.
- Nucleophilic attack of the 3’ OH end of the nick onto the recently added phosphate group. - Displacement of AMP/NAD+ seals the nick.
Problem: Helicase introduces positive supercoiling and products coil.
What is the solution? Is ATP involved?
Solution: Topoisomerase II
ATP is involved.
Passing of one portion of the double helix, through the next, with the help of ATP.
Each turn unwound during replication is 1 positive supercoil.
How is replication stopped in DNA replication?
DNA replication stops with the help of Tus proteins.
Tus Proteins (terminus utilization substances) bind to Ter sequences (20BP-long sequences that traps replication forks).
This will stall the replication, ending synthesis.
This is all mainly in circular DNA in bacteria.
What is the orientation of circular DNA after it has been replicated? What needs to happen afterwards?
Update, also in linear DNA
The two circular DNA molecules will be intertwined with each other, known as catenated chromosomes.
It separates the two, you need Topoisomerase IV.
What happens if DNA Polymerase III encounters an RNA Polymerase?
A DNA strand might be coding for protein the same time it is being replicated.
In this case, DNA Polymerase may run into RNA Polymerase, and it will cause both complexes to dissociated from the DNA strand.
But DNA Polymerase being the more predominant one, will bind to DNA again, using the mRNA placed from RNA Polymerase as it’s “primer” and continue to synthesis DNA.
