Ch 12 DNA Replication and Recombination
What are the three major requirements of replication?
(1) A single-stranded DNA template. (2) Nucleotide substrates for synthesis of the new polynucleotide strand. (3) Enzymes and other proteins associated with replication to assemble the nucleotide substrates into a new DNA molecule.
In what ways is eukaryotic replication different from bacterial replication?
(1) Eukaryotic DNA replication has multiple origins of replications per chromosome. (2) It has several different DNA polymerases with different functions. (3) Immediately following DNA replication, assembly of nucleosomes takes place.
What three mechanisms ensure the accuracy of replication in bacteria?
(1) Highly accurate nucleotide selection by the DNA polymerases when pairing bases. (2) The proofreading function of DNA polymerase, which removes incorrectly inserted bases. (3) A mismatch repair apparatus that repairs mistakes after replication is complete.
What are some of the enzymes taking part in recombination in E. coli ?
(1) RecBCD protein (2) RecA protein (3) RuvA and RuvB (4) RuvC protein (5) DNA ligase
In what ways is eukaryotic replication similar to bacterial replication?
(1) Semi-conservative replication. (2) Replication origins serve as starting points for replication. (3) Short segments of RNA called primers provide a 3'-OH for DNA polymerases to begin synthesis of the new strands. (4) Synthesis occurs in a 5' to 3' direction. (5) The template strand is read in a 3' to 5' direction. (6) Deoxyribonucleoside triphosphates are the substrates. (7) Replication is continuous on the leading strand and discontinuous on the lagging strand.
How did Meselson and Stahl demonstrate that replication in E. coli takes place in a semiconservative manner?
1. Grew E.coli in 15N medium. 2. Then shifted the bacteria to a 14N medium, DNA was isolated at different times corresponding to replication cycles 3. After one replication cycle, the DNA was all of intermediate density (ruled out the conservative replication model) 4. After two replication cycles, two bands of DNA were seen, one of intermediate density and one of light density (proof of semiconservative model and ruled out dispersive)
What would be the effect on DNA replication of mutations that destroyed 5' --> 3' polymerase activity in DNA polymerase I?
DNA polymerase I would be unable to synthesize new DNA strands if the 5'--> 3' polymerase activity was destroyed. RNA primers could be removed by DNA polymerase I using the 5'-->3' exonuclease activity, but could not be replaced by DNA polymerase I.
What similarities and differences exist in the enzymatic activities of DNA polymerases I, II, and III
Each of the three DNA polymerases has a 5' to 3' polymerase activity. They differ in their exonuclease activities. DNA polymerase I has both a 3' to 5' and a 5' to 3' exonuclease activity. DNA polymerase II and DNA polymerase III have only a 3' to 5' exonuclease activity.
A bacterium synthesizes DNA at each replication fork at a rate of 1000 nucleotides per second. If this bacterium completely replicates its circular chromosome by theta replication in 30 minutes, how many base pairs of DNA will its chromosome contain?
Each replication complex is synthesizing DNA at each fork at a rate of 1000 nucleotides per second. So for each second, 2000 nucleotides are being synthesized by both forks (1000 nucleotides / second × 2 forks = 2000 nucleotides / second) or 120,000 nucleotides per minute. If the bacterium requires 30 minutes to replicate its chromosome, then the size of the chromosome is 3,600,00 nucleotides (120,000 nucleotides / minute × 30 minutes = 3,600,000).
Suppose a future scientist explores a distant planet and discovers a novel form of double-stranded nucleic acid. When this nucleic acid is exposed to DNA polymerases from E. coli, replication takes place continuously on both strands. What conclusion can you make about the structure of this novel nucleic acid?
Each strand of the novel double-stranded nucleic acid must be oriented parallel to the other, as opposed to the antiparallel nature of earthly double-stranded DNA. Replication by E. coli DNA polymerases can only proceed continuously in a 5' to 3' direction, which requires the template to be read in a 3' to 5' direction. If replication is continuous on both strands, the two strands must have the same direction and be parallel.
What is semiconservative replication?
In semiconservative replication, the original two strands of the double helix serve as templates for new strands of DNA. When replication is complete, two double-stranded DNA molecules will be present. Each will consist of one original template strand and one newly synthesized strand that is complementary to the template.
Phosphorous is required to synthesize the deoxyribonucleoside triphosphates used in DNA replication. A geneticist grows some E. coli in a medium containing nonradioactive phosphorous for many generations. A sample of the bacteria is then transferred to a medium that contains a radioactive isotope of phosphorus (32P). Samples of the bacteria are removed immediately after the transfer and after one and two rounds of replication. What will be the distribution of radioactivity in the DNA of the bacteria in each sample? Will radioactivity be detected in neither, one, or both strands of the DNA?
In the initial sample removed immediately after transfer, very little if any 32P should be incorporated into the DNA since replication in the medium containing 32P has not yet occurred. After one round of replication in the 32P containing medium, one strand of each newly synthesized DNA molecule will contain 32P, while the other strand will contain only nonradioactive phosphorous. After two rounds of replication in the 32P containing medium, 50% of the DNA molecules will have 32P in both strands while the remaining 50% will contain 32P in one strand and nonradioactive phosphorous in the other strand.
How does replication licensing ensure that DNA is replicated only once at each origin per cell cycle?
Initiation can occur only at replication origins to which replication licensing factor (RPF) has bound. Shortly after the completion of mitosis, RPF binds the origin during G1 and is removed by the replication machinery during S phase.
Function of RuvC protein in E. coli
It is resolvase, , a protein that resolves the Holliday structure by cleavage of the DNA
What would be the effect on DNA replication of mutations that destroyed 5' --> 3' exonuclease activity in DNA polymerase I?
Loss of the 5' -->3' exonuclease activity would result in the RNA primers used to initiate replication not being removed by DNA polymerase I.
If the gene for primase were mutated so that no functional primase was produced, what would be the effect on theta replication?
Primase is required for replication initiation in theta form replication. If primase is nonfunctional then replication initiation would not take place resulting in no replication occurring.
Why is primase required for replication?
Primase synthesizes the short RNA molecules, or primers, that provide a 3'-OH to which DNA polymerase can attach deoxyribonucleotides in replication initiation. The DNA polymerases require a 3'-OH to which they add nucleotides, and therefore they cannot initiate replication. Primase does not have this requirement.
If the gene for primase were mutated so that no functional primase was produced, what would be the effect on rolling-circle replication?
Rolling-circle replication does not require primase. A single-stranded break within one strand provides a 3 OH group to which nucleotides can be added so rolling circle replication could occur without a functional primase
Explanation of Holliday model of homologous recombination
Single strand breaks in two adjacent DNA molecules allows for the free ends of the two strands to invade the other DNA molecule. The invading strand joins to the broken end of the other strand displacing complementary base pairs, which allows for the formation of a cross bridge. After formation, the cross bridge can move along the DNA molecules in a process called branch migration. The migration creates a structure called the Holliday intermediate. The cleavage might take place to generate recombinant DNA molecules. Resolution of the Holliday junction by cleavage can result either in spliced recombinants or patched recombinants.
The enzyme that replicated telomeres
Telomerase
What would be the effect on DNA replication of mutations that destroyed 3' --> 5' exonuclease activity in DNA polymerase I?
The 3' --> 5' exonuclease activity is important for proofreading newly synthesized DNA. If the activity is nonfunctional, then the fidelity of replication by DNA polymerase I will decrease, resulting in more misincorporated bases in the DNA.
How are telomeres replicated?
The RNA molecule in telomerase serves as a template. It moves along the DNA and adds nucleotides to the 3' end. After the 3' end has been extended, the 5' end of the DNA can be extended as well, possibly by lagging strand synthesis of a DNA polymerase using the extended 3' end as a template. DNA replication generates a 3' overhang. Part of the RNA sequence within telomerase is complementary to the overhang. Telomerase RNA sequence pairs with the 3' overhang and serves as a template for the addition of DNA nucleotides to the 3' end of the DNA molecule, which serves to extend the 3' end of the chromosome.Additional nucleotides to the 5' end are added by DNA synthesis using a DNA polymerase with priming by primase
Another name for DNA gyrase
Topoisomerase II
How would DNA replication be affected in a cell that is lacking topoisomerase?
Topoisomerase II or gyrase reduces the positive supercoiling or torsional strain that develops ahead of the replication fork due to the unwinding of the double helix. If the topoisomerase activity was lacking, then the torsional strain would continue to increase, making it more difficult to unwind the double helix. Ultimately, the increasing strain would lead to an inhibition of the replication fork movement
What is telomerase?
a ribonucleoprotein that consists of both protein and a RNA molecule that is complementary to the 3' end of the DNA of a eukaryotic chromosome. The RNA molecule also serves as a template for the addition of nucleotides to the 3' end.
Function of RecA protein in E. coli
allows a single strand to invade a double-stranded DNA
Initiator proteins function in bacterial replication
bind to the replication origin and unwind short regions of DNA
Function of DNA polymerase I in bacterial cells
carries out proofreading. It also removes and replaces the RNA primers used to initiate DNA synthesis.
Function of DNA ligase (in bacterial replication)
connects Okazaki fragments by sealing nicks in the sugar phosphate backbone
What substrates are used in the DNA synthesis reaction?
deoxyadenosine triphosphate (dATP) deoxyguanosine triphosphate (dGTP) deoxycytosine triphosphate (dCTP) deoxythymidine triphosphate(dTTP)
Function of DNA polymerase II in bacterial cells
functions as a DNA repair polymerase. It restarts replication after DNA damage has halted replication. It has proofreading activity
Function of DNA polymerase III (in bacterial replication)
primary replication polymerase elongates a new nucleotide strand from the 3'-OH of the primer
Function of RuvA and RuvB protein in E. coli
promote branch migration during homologous recombination
Function of DNA gyrase (in bacterial replication)
reduces DNA supercoiling and torsional strain that is created ahead of the replication fork by making double-stranded breaks in the DNA and passing another segment of the helix through the break before resealing it
Function of DNA ligase in E. coli
repairs nicks or cuts in the DNA generated during recombination
Single stranded binding protein (SSBB protein) function in bacterial replication
stabilizes single-stranded DNA prior to replication by binding to it, thus preventing the DNA from pairing with complementary sequences
Function of DNA primase (in bacterial replication)
synthesizes the RNA primers that provide the 3'-OH group needed for DNA polymerase III to initiate DNA synthesis
What are telomeres?
the caps at the end of each strand of DNA that protect chromosomes from degradation
Function of DNA polymerase III in bacterial cells
the primary replication enzyme and also has a proofreading function in replication
What is gene conversion? How does it arise?
the process by which one DNA sequence replaces a homologous sequence such that the sequences become identical after the conversion event. Its one of the two mechanisms of homologous recombination and involves the unidirectional transfer of genetic material from a 'donor' sequence to a highly homologous 'acceptor'.
Function of RecBCD protein in E. coli
unwinds double-stranded DNA and can cleave nucleotide strands
Function of DNA helicase (in bacterial replication)
unwinds the double-helix by breaking the hydrogen bonding between the two strands at the replication fork
