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.
Question: How do the mechanisms of prokaryotic and eukaryotic DNA replication compare?
Answer: There are remarkable similarities between prokaryotic and eukaryotic DNA replication mechanisms, but eukaryotic replication is slower due to more complex DNA structures (chromatin).
Question: Why are eukaryotic replication forks slower than prokaryotic replication forks?
Answer: Eukaryotic replication forks are slower due to the complexity of eukaryotic DNA structures (chromatin).
Question: At what speed do eukaryotic replication forks move?
Answer: Eukaryotic replication forks move at about 50 bp per second.
Question: Why are multiple replication forks required in eukaryotic DNA replication?
Answer: Due to the large size of eukaryotic genomes, 50,000 – 100,000 replication forks are required per mammalian cell.
Question: How do replicons initiate during eukaryotic DNA replication?
Answer: In eukaryotes, clusters of about 20-50 replicons initiate simultaneously at defined times during S-phase, based on accessibility to initiation factors (mitogens).
Question: What are the characteristics of the DNA replicated in early and late S-phase clusters in eukaryotes?
Answer:
Early S-phase clusters replicate euchromatin, which is transcriptionally active DNA.
Late S-phase clusters replicate heterochromatin, which is transcriptionally silent (ish) DNA.
Question: What regions of DNA are replicated last in eukaryotic DNA replication?
Answer: Centromeric and telomeric DNA are replicated last in eukaryotic DNA replication.
Question: What is the minimum DNA sequence length for supporting replication in yeast?
Answer: The minimum length of a DNA molecule that will support replication in yeast is 11 bp, with the sequence:
[A/T]TTTAT[A/G]TTT[A/T].
Question: What role does the Origin Replication Complex (ORC) play in DNA replication in yeast?
Answer: The Origin Replication Complex (ORC) binds to the replication origin sequence and is activated by cyclin-dependent kinases (CDKs). It facilitates and initiates the opening of the DNA duplex.
Question: What is known about the Origin Replication Complex (ORC) in mammals?
Answer: Defined ORCs have not been isolated in mammals, and it is believed that replication may initiate randomly in areas of repetitive DNA sequences.
Question: How is the initiation of eukaryotic DNA replication regulated?
[how does it initiate?
what regulates it
]
initiates once per cell to ensure full and controlled replication prior to cell division.
This is regulated by a protein licensing factor complex, which is inactivated after use and can only access the nucleus when the nuclear envelope dissolves during mitosis.
Question: What are the 4 key steps in the initiation of eukaryotic DNA replication?
Answer: The steps for initiating eukaryotic DNA replication are:
Identify the origin (binding of ORC).
Set up (binding of initiation factors).
Check the conditions.
Initiate the process and proceed with DNA replication.
Question: Why can’t the ends of chromosomes be replicated by semi-discontinuous replication?
Answer: The ends of chromosomes cannot be replicated by semi-discontinuous replication because there is no DNA to elongate once the RNA primer is removed from the 5’ end of the lagging strand, which could lead to the loss of genetic material.
Question: How do eukaryotic chromosomes solve the problem of replicating the ends (telomeres)?
[what does it have?,
which end hangs over the other?,
which enzyme is used?,
what does the enzyme used do ?
]
Answer: Eukaryotic chromosomes have a non-coding repeat sequence (TTAGGG) at the ends (telomeres).
The 3’-end overhangs the 5’-end, and the enzyme telomerase uses short RNA molecules that are complementary to this sequence to add repeats to the 3’-end overhang.
Question: What is the role of telomerase in replicating chromosome ends?
[what does it associate with?
what does ^ this act as?
]
Answer: Telomerase associates with short RNA molecules that are partially complementary to the telomere repeat sequence (TTAGGG).
The RNA acts as a template for the addition of the repeats to the 3’-end overhang.
Question: How is the complementary strand synthesized after the telomere repeats are added?
Answer: The complementary strand is synthesized by normal lagging strand synthesis, leaving the 3’-overhang intact.
Question: What happens to telomerase activity in somatic cells, and what is the consequence?
Answer: Telomerase activity is repressed in somatic cells, leading to a gradual loss of DNA and shortening of chromosomes, which may contribute to aging-related problems.
LOOK AT Meselson and Stahl experiments (1958)
LOOK AT Meselson and Stahl experiments (1958)
