Ch. 8a DNA Replication (Exam 2) Flashcards
What is the formal name for the synthesis of DNA?
DNA replication
What is the result of the conservative replication mechanism?
One DNA molecule w/ 2 OG strands and another w/ completely new DNA
What is the result of the semi-conservative mechanism?
Two molecules each w/ one OG strand and one new strand
What is the result of the dispersive replication mechanism?
Two DNA molecules w/ a patchwork of OG and new DNA
Who confirmed that DNA replication was semi-conservative?
Meselson & Stahl
How did Meselson & Stahl determine that DNA replication was semi-conservative?
They set up an experiment using N-15 (heavy) & N-14 (light) isotopes
In which phase of the cell cycle does replication take place for eukaryotes?
S
What happens in the G1 phase?
Everything expect for DNA is doubled
What phase starts the cell cycle?
G1
For what 2 reasons is there a checkpoint between the G1 and S phases?
1) Damaged DNA
2) Too little growth
Define replicon.
Genomic unit in which DNA is replicated
In which phase does the first replicon activate?
S
What is the origin of replication?
Site in DNA sequence where replication occurs
What structures form at the origin of replication?
2 replication forks
Which way does replication occur?
Replication is bi-directional
How do eukaryotic chromosomes differ from prokaryotic ones with respect to their origins of replication?
Eukaryotes have multiple
What is a replication bubble?
Bulge in DNA resulting from replication
Generally describe the formation of replication bubbles in two steps starting with the formation of the replication forks.
1) Replication forks form at both sides of replication origin
2) Bubbles form along molecule as it replicates in opposite directions.
When do replication bubbles stop growing?
When the bubbles join together at the end of replication
When do replication bubbles stop growing?
When they join together at the end of replication
Bi-directional replication of the circular bacterial chromosome begins at this single origin of replication.
What is OriC?
Why do eukaryotic chromosomes have multiple origins of replication?
Eukaryotic chromosomes are huge and take time to replicate
In order for the initiation of replication to occur, does DNA need to be supercoiled or relaxed?
Relaxed
What general enzyme is responsible for the alteration of chromosomal structure?
Topoisomerase
How do DNA topoisomerases solve the topological issues associated with replication?
They introduce temporary single- or double-stranded breaks in DNA
What topological issues arise in replication?
DNA is being unwound, which messes with its structure and ultimately its various functions (recombination, transcription, etc.)
What is initiation in terms of replication?
The start of replication
Initiation involves recognition of an origin by a…
Protein complex
What 3 things must happen to the parental strands before replication occurs?
Separation, strand stabilization, and forming a replication bubbles
What is a replisome?
Protein complex associated with the structure of DNA at the replication fork
How does the replisome aid in replication?
The replisome moves along the DNA, unwinding it and synthesizing daughter strands
What is meant by the term “de novo” when referring to the replisome?
It reassembles at the origin for each replication cycle
Can the replisome exist as independent protein units?
No
Why does the replisome have to assemble at the origin of each replication cycle?
All parts of the protein complex have to work together to function
What is helicase?
The enzyme that separates DNA strands using energy from ATP
What protein is responsible for maintaining the integrity of separated DNA strands?
Single-stranded DNA binding protein
What does single-stranded DNA binding protein do to help the DNA polymerases?
It binds to the single strands very well to allow the DNA polymerases to bind to their substrate
What are three key enzymes that you’ll find in a replisome?
1) Helicase
2) Primase
3) DNA polymerase
To reiterate, DNA replication as we know it is… (HINT: replication mechanism)
Semi-conservative
What does DNA polymerase do?
Synthesizes 5’ - 3’ daughter strands
What synthetic activity do all prokaryotic and eukaryotic DNA polymerases share?
Antiparallel synthesis from 5’ to 3’ from a 3’ to 5’ template
Although there are many DNA polymerases, there is only { } replication enzyme.
One
Which DNA polymerase is the replication polymerase responsible for de novo synthesis of new DNA strands?
DNA polymerase III
Which 2 DNA polymerase classes are required for nuclear replication?
Delta and epsilon
What are holoenzymes?
Replication polymerases incorporated into large protein assemblies
If the DNA-synthesizing subunit is only one of several functions of the holoenzyme, what functions do other subunits serve?
Making sure that template replication is accurate e.g. proofreading
Which end of the parental strand does DNA polymerase add nucleotides to?
3’ end
DNA polymerase has both polymerase (synthesis) and { } activity (HINT: the blank refers to its enzymatic, degradative properties).
Nuclease
Nuclease has the ability to break this type of bond between nucleotides…
What is phosphodiester?
DNA polymerase I uses this enzyme to perform nick translation…
5’-3’ nuclease
What is the function of 5’-3’ exonuclease?
To remove the RNA primers
What are the 2 primary functions of DNA polymerase I?
Repair damaged DNA and remove RNA primers, replacing them with DNA
What is the function of 3’-5’ exonuclease?
To remove incorrectly paired bases at the 3’ end, effectively proofreading
In which direction is DNA synthesized?
5’ to 3’
In which direction is the DNA template strand?
3’ to 5’
The leading strand synthesizes…
Continuously from 5’ to 3’ toward the replication fork
The lagging strand quite literally…
Lags
Why does the lagging strand lag?
DNA is antiparallel, and there is only one free 3’ end in synthesis, but it is already occupied with synthesizing the leading strand
What does it mean for the lagging strand to lag?
This strand is synthesized away from the replication fork, so it is synthesized in fragments
What is the official name for the fragments created by the lagging strand?
Okazaki fragments
What is a primer?
A short nucleic acid (RNA) sequence with an open 3’ OH end that serves as the starting point for DNA synthesis
What is RNA primase?
A special RNA polymerase that synthesizes primers
What enzyme extends the primer?
DNA polymerase
In terms of the initiation of priming activity, how does the lagging strand differ from the leading strand?
The leading strand only requires one initiation event to get its primers going while the lagging strand requires multiple initiation events
Why does the lagging strand require multiple initiation events for its priming activity?
The Okazaki fragments require the primer activity to start and restart (de novo) for each strand
What is the function of DNA ligase?
To form phosphodiester bonds between Okazaki fragments
What intermediate molecule works alongside DNA ligase in forming the phosphodiester bonds?
Adenosine monophosphate (AMP)
What is termination?
The completed replication of the parent molecule
When does bidirectional replication terminate?
When two replication forks meet halfway around the circle
What are the sites that cause termination called?
Ter sites
How does the Meselson-Stahl experiment start?
The N-15 (heavy) isotope is used to label the parental DNA molecule, so it is now a duplex of “heavy” strands
After the N-15 isotope labels the parental DNA molecule, what happens next in the Meselson-Stahl experiment?
The molecule spends a generation in the N-14 (light) isotope
What is the result of the parental DNA molecule (N-15-labeled) growing a generation in the N-14 isotope?
The parental molecule spawns a hybrid consisting of one N-14 strand and one N-15 strand
In the Meselson-Stahl experiment, the N-14, 15 hybrid strand is cultivated in more N-14 isotope for another generation of replication. What is the result?
Semi-conservative replication such that the N-14 strand is paired with another N-14 strand, and the N-15 strand is paired with an N-14 as well
