Week 27 +28/ DNA Replication Flashcards
Question: What are the three models of DNA replication?
[explain each]
Semiconservative replication – Produces two DNA duplexes, each containing one old strand and one new strand.
Conservative replication – Produces two DNA duplexes: one with both old strands and one with both new strands.
Dispersive replication – Produces two DNA duplexes, each with strands that are a mix of old and new DNA.
Question: Where does DNA replication initiate?
[where does it initiate , what does initiation create]
Answer: DNA replication initiates at an ‘origin of replication’, creating two replication forks.
Question: How does DNA replication compare between prokaryotes and eukaryotes?
Answer: The process is broadly similar but more complex in eukaryotes due to their larger genome.
Question: How many origins of replication do prokaryotes and eukaryotes have?
Answer: Prokaryotes have a single origin, while eukaryotes have multiple origins to accommodate their larger genome.
Question: What is a replication fork?
Answer: A replication fork is the branch point in a replication eye where DNA synthesis occurs.
Question: What is a replication bubble, and[ how many replication forks can it have?]
Answer: A replication bubble is a region where DNA is unwound for replication. It can have one replication fork (unidirectional replication) or two replication forks (bidirectional replication).
Question: Is DNA replication usually unidirectional or bidirectional?
Answer: DNA replication is almost always bidirectional.
Question: How many origins of replication do prokaryotic and bacteriophage DNAs have?
Answer: Prokaryotic and bacteriophage DNAs have a single origin of replication, where DNA synthesis is initiated.
Question: What are the 8 steps involved in replication initiation in prokaryotes?
Answer:
DnaA proteins bind to four 9-bp repeats at the origin of replication.
More DnaA proteins join cooperatively, forming a DnaA barrel.
The DnaA barrel causes torsional stress, opening the nearby AT-rich region (easier to pull apart due to fewer hydrogen bonds).
Opening of DNA creates two replication forks — the replication process begins.
DnaB helicase is recruited; it unwinds DNA by breaking hydrogen bonds.
Single-stranded binding proteins (SSBs) attach to the open strands, keeping them apart and protecting them.
DnaA proteins bind to four copies of a 9-bp sequence in the origin of replication.
Once all binding sites are occupied, DnaA proteins cooperatively recruit more DnaA proteins, forming a DnaA barrel.
The DnaA barrel generates torsional stress, opening a local AT-rich region of the DNA.
This leads to the generation of a pair of replication forks, initiating DNA replication.
DnaB helicase is recruited to the replication fork to begin the formation of the pre-priming complex.
DnaB breaks the hydrogen bonds between base pairs, unwinding the DNA.
The open DNA strands are covered with SSBs (single-stranded binding proteins), which prevent the strands from re-annealing and protect the DNA from free radicals and nucleases.
With these processes complete, DNA replication initiation is finished, and the next phase, elongation, begins.
Question: What are the steps involved in replication initiation in prokaryotes after the formation of the replication forks? [4]
Answer:
DnaB helicase is recruited to the replication fork to begin the formation of the pre-priming complex.
DnaB breaks the hydrogen bonds between base pairs, unwinding the DNA.
The open DNA strands are covered with SSBs (single-stranded binding proteins), which prevent the strands from re-annealing and protect the DNA from free radicals and nucleases.
With these processes complete, DNA replication initiation is finished, and the next phase, elongation, begins.
Question: How does the polymerization reaction occur during DNA replication? [3]
[what is polymerization?
which enzymes are used?
which direction is the DNA generated?
which direction does the polymerase move?
]
Answer:
The synthesis of the new DNA strand
is carried out by DNA-dependent DNA polymerase enzymes.
DNA is generated in the 5’ to 3’ direction.
The polymerase moves along the template strand in the 3’ to 5’ direction.
Question: What are the steps involved in prokaryotic elongation? [3]
[where does primase enzyme do? (what does it form)
what do Single-strand binding proteins do?
what do the The DNA polymerase III holoenzyme do?]
Answer:
Primase enzyme (DnaG) binds near the helicase and starts synthesizing the RNA primer on the leading strand, forming the primosome.
Single-strand binding proteins (SSBs) stabilize the lagging strand.
The DNA polymerase III holoenzyme clamps onto the leading strand and begins synthesizing DNA.
Question: What happens during semi-discontinuous replication? [4]
[in which direction does DNA synthesis carried out by DNA polymerase?
which nucleotide does polymerase insert first?
which direction is the template used?
whats is the lagging strand generated from?
what is the lagging strand synthesised opposite to ?
]
DNA synthesis is carried out by DNA polymerase in the 5’ to 3’ direction.
The polymerase inserts the 5’ nucleotide first and extends towards the 3’ end.
The template DNA is always read in the 3’ to 5’ direction.
The lagging strand is synthesized in the opposite direction to the movement of the replication fork, through the creation of multiple Okazaki fragments.
Q1: What enzyme starts DNA replication by making a short RNA primer?
Q2: Why is the RNA primer important?
Q3: How is the RNA primer removed?
Q4: How are both strands of DNA replicated at the same time?
Q5: How is the leading strand replicated?
Q6: How is the lagging strand replicated?
Q7: Why is the lagging strand looped?
Q8: What prevents DNA from tangling ahead of the fork?
Q9: What joins the Okazaki fragments together?
Q10: What is the name of the entire complex doing replication?
Question: How are Okazaki fragments handled during DNA replication?
A1: Primase synthesizes the primer in E. coli.
A2: It provides a starting point and allows for proofreading of the new DNA strand.
A3: By the exonuclease activity of DNA polymerase I.
A:4 Two DNA polymerase enzymes are tethered together in the replisome.
A5: Continuously in the 5’ to 3’ direction.
A6: Discontinuously in the 5’ to 3’ direction as Okazaki fragments.
A7: So both polymerases can move in the same direction.
A8: Topoisomerase enzymes.
A9: The ligase enzyme during ligation.
A10: The replisome.
Answer:
DNA replication starts with a short RNA primer, allowing proofreading of the newly synthesized strand.
The primase enzyme synthesizes the primer in E. coli.
The primer is removed by the exonuclease activity of the polymerase complex.
To replicate both strands simultaneously, two DNA polymerase enzymes are tethered together:
One replicates the leading strand continuously in the 5’ to 3’ direction.
One replicates the lagging strand discontinuously, also in the 5’ to 3’ direction.
The lagging strand is looped over the top of the replisome so that both polymerases move in the same direction.
Topoisomerase enzymes work ahead of the replisome to prevent tangling.
The Okazaki fragments are joined by a ligase enzyme during ligation.
DNA polymerase III is replaced by DNA polymerase I (Pol I), which removes the RNA primer on the Okazaki fragment before ligation.
This whole process is called the replisome.
Question: Why do replication forks face a topological problem during DNA replication?
Answer: Replication forks can only progress a short distance before encountering a topological problem due to the over-winding of the DNA ahead of the replication fork.
Question: How does the double helix handle the over-winding issue during replication?
[what does it require]
Answer: The double helix requires rotation as it is opened to stop over-winding ahead of the replication fork.
Question: What is the role of topoisomerases in DNA replication?
Answer: Topoisomerases alleviate the topological problems during replication:
Type I topoisomerase introduces a break in one strand, passes the other strand through, and reseals the break.
Type II topoisomerase breaks both strands, passes a double helix through the gap, and reseals the break.
Question: How do topoisomerases prevent losing DNA ends during the breakage process?
Answer: The breaks in DNA are covalently attached to the topoisomerase enzymes, preventing the loss of the DNA ends.
Question: Where do the two replicons meet during DNA replication in E. coli?
Answer: In E. coli, the two replicons meet 180° away from the origin of replication.
Question: How is the meeting of the two replicons regulated in DNA replication?
Answer: A regulatory mechanism ensures that the replicons meet at a specific point. If one gets there first, it will wait for the other replicon to arrive before signaling that DNA replication is complete.
Question: What signals that the replicon is approaching the stop sequence during DNA replication?
Answer: Specific terminator sequences signal that the replicon is approaching the stop sequence.
Question: How does the replicon respond when it encounters a transcription bubble during replication?
Answer: If the replicon meets a transcription bubble (mRNA synthesis), it will wait and not overtake.
Question: What happens to the newly generated daughter DNA molecules?
Answer: Once the two daughter DNA molecules are generated, they are interlinked (catenated), and topoisomerase enzymes unlink them.
Question: How are the separated DNA molecules handled in preparation for cell division?
Answer: The separated DNA molecules are segregated, awaiting cell division, where each new cell will receive one DNA molecule.
