Chapter 9: DNA Replication and Recombination Flashcards
What occurs during the S phase?
The DNA duplicates through the production of a sister chromatid
What is conserved replication?
- The entire double-stranded DNA molecule serves as a template for a whole new molecule of DNA
- The original DNA molecule is fully conserved during replication
What is dispersive replication?
Both nucleotide strands break down (disperse) into fragments, which serve as templates for the synthesis of new DNA fragments, and then somehow reassemble into two complete DNA molecules
What results from dispersive replication?
Each resulting DNA molecule contains interspersed fragments of old and new DNA; none of the original molecule is conserved
What is semiconservative replication?
- The two nucleotide strands unwind, and each serves as a template for a new DNA molecule
- The template is the original strand from which the synthesized strand is based on
What did Meselson and Stahl discover?
Experiment to determine which proposed model of replication was correct
What experimental method did Meselson and Stahl utilize? What occurs?
- Equilibrium density gradient centrifugation
- Heavy DNA (with 15N) will move toward the bottom
- Light DNA (with 14N) will remain closer to the top
What occurred in the Meselson and Stahl experiment when E. coli contained in 15N medium was transferred to 14N medium and replicated once?
The DNA appeared at a single band at intermediate weight
What occurred in the Meselson and Stahl experiment when E. coli contained in 15N medium was transferred to 14N medium and replicated twice?
DNA appeared as two bands, one light and the other intermediate in weight
What results from the Meselson and Stahl experiment were inconsistent with conservative replication?
Conservative replication predicts one heavy band (original DNA) and one light band (new DNA molecules)
What organisms undergo theta replication?
Common in E. coli and other organisms possessing circular DNA
What occurs at an origin of replication?
- Enzymes allow the unwinding of dsDNA
- Produces a replication bubble, usually having a replication fork at each end
When does bidirectional replication occur?
- When there are two replication forks, one at each end of the replication bubble, proceeding outward in both directions
- Simultaneously unwinding and replicating until the DNA eventually meet
When does unidirectional replication occur?
- Occurs when a single replication fork is present
- Proceeds around the entire circle to produce two complete circular DNA molecules, each consisting of one old and one new nucleotide strand
Which organisms undergo rolling-circle replication?
- Some viruses
- F factor of E. coli
How many replication bubbles and forks are formed in the rolling-circle replication?
There are NO replication bubbles or forks
What are the two strategies that permit rapid reading and replication of the Eukaryotic genome?
1) Presence of extremely powerful and rapid polymerase enzymes
2) Multiple origins of replication are present
Why are multiple origins of replication necessary for Eukaryotes?
- Because as the genome size increases, the speed of the enzyme is not sufficient
- As the speed of the enzyme increases, the likelihood of an error increases as well
What increases with the number of origins of replication?
The average length of the replicon (DNA sequence synthesized)
During bidirectional replication, the forks of adjacent bubbles require _____.
fusion
What are the products of Eukaryotic DNA replication?
Two linear DNA molecules
What is the DNA template in theta, rolling-circle and linear Eukaryotic replication?
- Theta: circular
- Rolling-circle: circular
- Linear eukaryotic: linear
Does breakage of the nucleotide strand occur in theta, rolling-circle and linear Eukaryotic replication?
- Theta: no
- Rolling-circle: yes
- Linear eukaryotic: no
How many replicons are present in theta, rolling-circle and linear Eukaryotic replication?
- Theta: 1
- Rolling-circle: 1
- Linear eukaryotic: many
Does unidirectional or bidirectional replication occur in theta, rolling-circle and linear Eukaryotic replication?
- Theta: unidirectional or bidirectional
- Rolling-circle: unidirectional
- Linear eukaryotic: bidirectional
What products are synthesized from theta, rolling-circle and linear Eukaryotic replication?
- Theta: two circular molecules
- Rolling-circle: one circular molecule and one linear molecule that may circularize
- Linear eukaryotic: two linear molecules
The newly synthesized DNA strand is _______ and __________ to the template strand.
complementary, anti-parallel
What is new DNA synthesized from?
dNTPs
How does a phosphodiester bond form?
- 3’-OH group of the last nucleotide on the strand attacks the 5’-phosphate group of the incoming dNTP
- Two phosphates are cleaved off
- A phosphodiester bond forms between the nucleotides and phosphate ions are released
DNA synthesis takes place in the (same/opposite) direction on the two DNA template strands.
opposite
Where is DNA synthesis continuous? What is it? What is the exposed template?
- Leading strand
- Template exposed: 3’ –> 5’
Where is DNA synthesis discontinuous? What is it? What is the exposed template?
- Lagging strand
- Template exposed: 5’ –> 3’
- Okazaki fragments
A replication bubble requires a ____ on both sides.
fork
What occurs in terms of DNA synthesis on the leading template strand?
Proceeds continuously in the 5’ –> 3’ direction
What occurs in terms of DNA synthesis on the lagging template strand?
- DNA synthesis begins at the fork and proceeds in the direction opposite of unwinding, and eventually runs out of template
- DNA synthesis starts again, at each fork
What are Okazaki fragments?
Short fragments of DNA produced by discontinuous synthesis
Does DNA polymerase I possess:
- 5’ –> 3’ polymerization
- 3’ –> 5’ exonuclease
- 5’ –> 3’ exonuclease activity?
- Yes
- Yes
- Yes
Does DNA polymerase II possess:
- 5’ –> 3’ polymerization
- 3’ –> 5’ exonuclease
- 5’ –> 3’ exonuclease activity?
- Yes
- Yes
- No
Does DNA polymerase III possess:
- 5’ –> 3’ polymerization
- 3’ –> 5’ exonuclease
- 5’ –> 3’ exonuclease activity?
- Yes
- Yes
- No
Does DNA polymerase IV possess:
- 5’ –> 3’ polymerization
- 3’ –> 5’ exonuclease
- 5’ –> 3’ exonuclease activity?
- Yes
- No
- No
Does DNA polymerase V possess:
- 5’ –> 3’ polymerization
- 3’ –> 5’ exonuclease
- 5’ –> 3’ exonuclease activity?
- Yes
- No
- No
What is the function of DNA polymerase I?
- Removes and replaces primers with DNA
- Then, it uses its 5’ –> 3’ polymerase activity to replace the RNA nucleotides with DNA nucleotides
What is the function of DNA polymerase II?
- DNA repair
- Restarts replication after damaged DNA halts synthesis
What is the function of DNA polymerase III?
Elongates DNA from the 3’-OH group provided by the primer
What is the function of DNA polymerase IV?
DNA repair
What is the function of DNA polymerase V?
- DNA repair
- Translesion DNA synthesis
What allows most of the errors that arise in nucleotide selection to be correcting in proofreading?
Exonuclease activity
How does the exonuclease activity of DNA polymerase remove incorrectly paired nucleotides?
The 3’-OH mispaired nucleotide is not correctly position, and the DNA polymerase removes the incorrectly paired nucleotide
How does bacterial DNA replication being?
- Initiator proteins bind to the origin of replication
- Causes a short stretch of DNA to unwind
- Unwinding allows helicase and other single-strand binding proteins to attach to the single-stranded DNA
How does DNA helicase unwind DNA?
- By binding to the lagging-strand template at each replication fork and moving in the 5’ –> 3’ direction
- Breaks hydrogen bonds and moves the replication fork
What stabilizes the exposed single-stranded DNA in bacterial replication?
Single-strand-binding proteins
What is the function of DNA gyrase?
Relieves torsional strain that builds up ahead of the replication fork as a result of unwinding
How does DNA gyrase reduce torque?
- Makes a double-strand break in one segment of the DNA helix
- Passes another segment of the helix through the break
- Reseals the broken ends of the DNA (reduces supercoiling)
What kind of enzyme is DNA primase?
- RNA polymerase
- NOT a DNA polymerase
Why is DNA primase necessary?
- Since nucleotides may only be added at the 3’ end, but they must be attached to the primer
- RNA molecules may be added anywhere, without a free 3’ end
How does DNA primase add primers?
Synthesizes a short RNA primer to provide a free 3’-OH group for the attachment of DNA nucleotides
Where are primers required on the leading strand?
Only at the 5’ end of the newly synthesized strand
Where are primers required on the lagging strand?
A new primer must be generated at the beginning of each Okazaki fragment
What is the function of DNA ligase?
Joins Okazaki fragments by sealing breaks with a phosphodiester bond between the 5’-P group of the initial nucleotide added by DNA polymerase III and the 3’-OH group of the final nucleotide added by DNA polymerase I
What is the hypothesized model of DNA replication in E. coli?
- The two units of DNA polymerase III are connected
- DNA is moving, and enzymes are relatively fixed in space
- Lagging-strand forms a loop so that replication may take place on the two antiparallel DNA strands
What is the function of initiator protein?
Binds to origin and separates strands of DNA to initiate replication
What is the function of DNA helicase?
Unwinds DNA at replication fork
What is the function of single-strand-binding proteins?
Attach to single-stranded DNA and prevent secondary structures from forming
What is the function of DNA gyrase?
- Moves ahead of the replication fork, making and resealing breaks in the double-helical DNA
- Releases the torque that builds up as a result of unwinding at the replication fork
What is the function of DNA primase?
Synthesizes a short RNA primer to provide a 3’-OH group for the attachment of DNA nucleotides
What is the function of DNA polymerase III?
Elongates a new nucleotide strand from the 3’-OH provided by the primer
What is the function of DNA polymerase I?
Removes RNA primers and replaces them with DNA
What is the function of DNA ligase backbone?
Joins Okazaki fragments by resealing nicks in the sugar-phosphate of newly synthesized DNA
Does DNA polymerase alpha possess:
- 5’ –> 3’ polymerase activity
- 3’ –> 5’ exonuclease activity
- Yes
- No
Does DNA polymerase beta possess:
- 5’ –> 3’ polymerase activity
- 3’ –> 5’ exonuclease activity
- Yes
- No
Does DNA polymerase gamma possess:
- 5’ –> 3’ polymerase activity
- 3’ –> 5’ exonuclease activity
- Yes
- Yes
Does DNA polymerase delta possess:
- 5’ –> 3’ polymerase activity
- 3’ –> 5’ exonuclease activity
- Yes
- Yes
Does DNA polymerase epsilon possess:
- 5’ –> 3’ polymerase activity
- 3’ –> 5’ exonuclease activity
- Yes
- Yes
What is the function of DNA polymerase alpha?
- Initiation of nuclear DNA synthesis and DNA repair
- Has primase activity
What is the function of DNA polymerase beta?
- DNA repair
- Recombination of nuclear DNA
What is the function of DNA polymerase gamma?
Replication and repair of mitochondrial DNA
What is the function of DNA polymerase delta?
- Leading- and lagging-strand synthesis of nuclear DNA
- DNA repair
- Translesion DNA synthesis
What is the function of DNA polymerase epsilon?
Leading-strand synthesis
Why is DNA synthesis much simpler in Prokaryotes?
As they possess circular DNA
What is the consequence of telomeres on DNA synthesis?
- Telomeres fold on themselves to protect the chromosome from degradation
- But, at a certain point, there is no more 3’-end for the addition of nucleotides, resulting in their decrease in length
In the absence of special mechanisms, DNA replication in Eukaryotes would leave ________
gaps due to the removal of primers at the ends of chromosomes
Which cells express the enzyme telomerase?
- Specialized cells
- Germ cells and bone marrow cells
What is the function of telomerase?
- Responsible for the replication of chromosome ends (of a single strand)
- The complimentary strand is elongated after the removal of the telomerase
What did mice models expressing increased levels of telomerase cause?
Increased incidence of cancer
What occurs after one round of replication with conservative replication?
- 50% of the molecules would consist of the original DNA
- 50% of the molecules would consist of entirely new DNA
What occurs after two rounds of replication with conservative replication?
- 25% original DNA
- 75% entirely new DNA
How would the proportion of molecules with new DNA fluctuate, and the number of molecules with original DNA change with each round of replication in conservative replication?
- The proportion of molecules with new DNA increases
- The number of molecules with original DNA remains constant
How would the proportion of molecules with new DNA fluctuate with each round of replication in dispersive replication?
- Would always produce a hybrid molecule
- New DNA within the molecules would increase with each replication event
How many bands of DNA would be expected in Meselson and Stahl’s experiment after two rounds of conservative replication?
Two bands
Discontinuous replication is a result of which property of DNA? A) Complementary bases B) Charged phosphate groups C) Antiparallel nucleotide strands D) Five-carbon sugar
C) Antiparallel nucleotide strands
What are the four stages of replication?
- Initiation
- Unwinding
- Elongation
- Termination
Can helicase initiate the unwinding of double-stranded DNA?
No, initiator proteins first separate the DNA strands at the origin
Place the following components in the order in which they are first used in the course of replication:
- Helicase
- Single-strand-binding protein
- DNA gyrase
- Initiator proteins
1) Initiator proteins
2) Helicase
3) Single-strand-binding protein
4) DNA gyrase
Primers are synthesized where on the lagging strand?
A) Only at the 5’ end of the newly synthesized strand
B) Only at the 3’ end of the newly synthesized strand
C) At the beginning of every Okazaki fragment
D) At multiple places within an Okazaki fragment
C) At the beginning of every Okazaki fragment
After primers have been removed and replaced, the break in the sugar-phosphate linkage is sealed by ___________.
DNA ligase
Which bacterial enzyme removes the primers? A) Primase B) DNA polymerase I C) DNA polymerase III D) Ligase
B) DNA polymerase I
What does each active replication fork require? (5)
1) Helicase
2) Single-strand-binding proteins
3) Topoisomerase gyrase
4) Primase
5) DNA polymerase
What allows for accurate DNA replication?
- Precise nucleotide selection
- Proofreading
- Mismatch repair
What are some differences from prokaryotes in the genome structure of eukaryotic cells that affect how replication takes place?
- The size of the eukaryotic genome
- The linear structure of eukaryotic chromosomes
- The association of DNA with histone proteins
How does a cell ensure that replication is initiated at thousands of origins only once per cell cycle?
1) The origins are licensed and approved for replication (replication licensing factor)
2) The replication machinery initiates replication at each licensed origin
3) The licensing factor is removed as the replication forks move away from the origin
Which eukaryotic DNA polymerases carry out replication on the leading and lagging strands?
Alpha, delta, and epsilon
What does chromosome shortening mean?
That when an organism is reproduced, it would pass on shorter chromosomes than it inherited
Which end does telomerase elongate? How?
- The 3’ end of the chromosome
- Without the use of complementary DNA template
- Adds extra nucleotides to the G-rich DNA strand of the telomere
What would result if an organism’s telomerase were mutated and non-functional?
A) No DNA replication would take place
B) The DNA polymerase enzyme would stall at the telomere
C) Chromosomes would shorten each generation
D) RNA primers could not be removed
C) Chromosomes would shorten each generation
Why is recombination important?
To generate genetic variation
What would be the effect on DNA replication of mutations that destroyed 3’ → 5’ exonuclease activity?
More errors in replication
What would be the effect on DNA replication of mutations that destroyed 5’ → 3’ exonuclease activity?
Primers would not be removed
What would be the effect on DNA replication of mutations that destroyed 5’ → 3’ polymerase activity?
Primers that had been removed would not be replaced
In DNA replication
A) both strands replicate in the same direction
B) each strand replicates in a different direction
C) only one strand of DNA is used as a template
D) a single strand of DNA is copied to make two single strands of DNA
D) a single strand of DNA is copied to make two single strands of DNA
Semiconservative replication of DNA means
A) only one strand is used as a template
B) a double-stranded DNA is split into two single-stranded DNAs
C) only half the genes are copied into the new cells
D) each DNA made contains one old strand and one new strand of DNA
A) only one strand is used as a template
In the leading strand, DNA synthesis occurs ___________ the replication fork. In the lagging strand, DNA synthesis occurs ________________ the replication fork.
toward
away from
Primers are synthesized where on the leading strand?
A) Only at the 5′ end of the newly synthesized strand
B) Only at the 3′ end of the newly synthesized strand
C) At the beginning of every Okazaki fragment
D) At multiple places within an Okazaki fragment
A) Only at the 5′ end of the newly synthesized strand
Which mechanism requires the ability to distinguish between newly synthesized and template strands of DNA? A) Nucleotide selection B) DNA proofreading C) Mismatch repair D) All of the above
C) Mismatch repair
In comparison with prokaryotes, what are some differences in the genome structure of eukaryotic cells that affect how replication takes place?
The size of eukaryotic genomes, the linear structure of eukaryotic chromosomes, and the association of DNA with histone protein
Some of the eukaryotic DNA polymerases have a tendency to make errors in replication. Why would a cell use an error-prone DNA poly- merase instead of one that is more accurate?
Because error-prone DNA polymerases can bypass bulky lesions in the DNA helix that stall accurate, high- speed DNA polymerases
