When is DNA replicated?
replicated once every time a cell divides
In which direction does DNA synthesis occur?
5’ to 3’ direction by DNA polymerase
What are the 3 hypotheses of DNA replication?
- conservative
- semiconservative
- dispersive
Conservative Model of DNA replication
In this model the two parental DNA strands are back together after replication has occurred. That is, one daughter molecule contains both parental DNA strands, and the other daughter molecule contains DNA strands of all newly-synthesized material
Semiconservative Model of DNA replication
In this model the two parental DNA strands separate and each of those strands then serves as a template for the synthesis of a new DNA strand. The result is two DNA double helices, both of which consist of one parental and one new strand.
Dispersive Model of DNA replication
In this model the parental double helix is broken into double-stranded DNA segments that, as for the Conservative Model, act as templates for the synthesis of new double helix molecules. The segments then reassemble into complete DNA double helices, each with parental and progeny DNA segments interspersed.
Dispersive Model of DNA replication
In this model the parental double helix is broken into double-stranded DNA segments that, as for the Conservative Model, act as templates for the synthesis of new double helix molecules. The segments then reassemble into complete DNA double helices, each with parental and progeny DNA segments interspersed.
Who did what experiment to define the nature of DNA replication?
Meselson and Stahl Experiment
-used heavy isotopes (more dense) N15 that can incorporate itself into DNA without affecting DNA function
(DNA is normally made of N14)
*when bacteria are grown in a medium containing N15 their DNA is denser than bacteria that grow under normal conditions with N14
-DNA of different densities can be separated through centrifugation in a heavy salt (CsCl) density gradient. Heavy DNA towards the outer end> 14N-15N hybrid DNA> light DNA on the inner end
Setup:
- Grow E.coli cells in a medium with 15N as sole source of nitrogen. Collect sample and purify DNA. (15N-15N heavy DNA)
- Transfer cells to medium containing 14N. After cells divide once, collect sample and purify DNA. (15N-14N hybrid DNA)
- After cells have divided a second time in 14N medium, collect sample and purify DNA. (15N-14N hybrid DNA and 14N-14N light DNA)
- Centrifuge the three samples and compare the locations of the DNA bands.
Who did what experiment to define the nature of DNA replication?
Meselson and Stahl Experiment
-used heavy isotopes (more dense) N15 that can incorporate itself into DNA without affecting DNA function
(DNA is normally made of N14)
*when bacteria are grown in a medium containing N15 their DNA is denser than bacteria that grow under normal conditions with N14
-DNA of different densities can be separated through centrifugation in a heavy salt (CsCl) density gradient. Heavy DNA towards the outer end> 14N-15N hybrid DNA> light DNA on the inner end
Setup:
- Grow E.coli cells in a medium with 15N as sole source of nitrogen. Collect sample and purify DNA. (15N-15N heavy DNA)
- Transfer cells to medium containing 14N. After cells divide once, collect sample and purify DNA. (15N-14N hybrid DNA)
- After cells have divided a second time in 14N medium, collect sample and purify DNA. (15N-14N hybrid DNA and 14N-14N light DNA)
- Centrifuge the three samples and compare the locations of the DNA bands.
How to analyze the results of the Meselson & Stahl experiment.
Meselson and Stahl tested the hypothesis of DNA replication. They cultured bacteria in a 15N medium. 15N is a heavy isotope of nitrogen so the DNA synthesized is of heavy density. They then shifted the bacteria to a 14N medium, DNA was isolated at different times corresponding to replication cycles 0, 1, and 2. After one replication cycle, the DNA was all of intermediate density. This rules out the conservative replication model, which predicts that both heavy density DNA and light density DNA will be present, but none of intermediate density will be present. This result is consistent with the semiconservative replication model, which predicts that all DNA molecules will consist of one 15N-labeled DNA strand and one 14N-labeled DNA strand. The result does not rule out the dispersive replication model, which also predicts that all DNA will be of intermediate density, consisting of interspersed double-stranded 15N-labeled and 14N-labeled segments.
After two replication cycles, two bands of DNA were seen, one of intermediate density and one of light density. This result is exactly what the semiconservative model predicts: half should be 15N-14N intermediate density DNA and half should be 14N-14N light density DNA. This result rules out the dispersive replication model, which predicts that after replication cycle 1, the DNA density of all DNA molecules will gradually become lower, so no intermediate density DNA should remain at replication cycle 2. The semiconservative model is correct.
How to analyze the results of the Meselson & Stahl experiment.
Meselson and Stahl tested the hypothesis of DNA replication. They cultured bacteria in a 15N medium. 15N is a heavy isotope of nitrogen so the DNA synthesized is of heavy density. They then shifted the bacteria to a 14N medium, DNA was isolated at different times corresponding to replication cycles 0, 1, and 2. After one replication cycle, the DNA was all of intermediate density. This rules out the conservative replication model, which predicts that both heavy density DNA and light density DNA will be present, but none of intermediate density will be present. This result is consistent with the semiconservative replication model, which predicts that all DNA molecules will consist of one 15N-labeled DNA strand and one 14N-labeled DNA strand. The result does not rule out the dispersive replication model, which also predicts that all DNA will be of intermediate density, consisting of interspersed double-stranded 15N-labeled and 14N-labeled segments.
After two replication cycles, two bands of DNA were seen, one of intermediate density and one of light density. This result is exactly what the semiconservative model predicts: half should be 15N-14N intermediate density DNA and half should be 14N-14N light density DNA. This result rules out the dispersive replication model, which predicts that after replication cycle 1, the DNA density of all DNA molecules will gradually become lower, so no intermediate density DNA should remain at replication cycle 2. The semiconservative model is correct.
What substrates are used for DNA replication?
- dNTPs: dGTP, dCTP, dATP, dTTP
2. Primer: a short sequence of nucleotides that is bp to the template with a 3’OH end of growing DNA
In a dNTP, which bond is high in energy?
the bond between the alpha and beta phosphate group. it breaks through hydrolysis which makes it an energetically favorable reaction
What is the chemistry of DNA synthesis?
A dNTP comes in to fill the spot at the end of the primer on the 3’OH side. The 3’OH of the primer bonds with the alpha phosphate group of the incoming dNTP and the dNTP base pairs with the template. Two phosphate groups of the dNTP are removed by PYROPHOSPHATES.
What is the chemistry of DNA synthesis?
A dNTP comes in to fill the spot at the end of the primer on the 3’OH side. The 3’OH of the primer bonds with the alpha phosphate group of the incoming dNTP and the dNTP base pairs with the template. Two phosphate groups of the dNTP are removed by PYROPHOSPHATES.
DNA Polymerase
- resembles a partially closed right hand
- DNA sits in the cleft of it
- has 3 domains:
1. palm (cleft where DNA sits)
2. thumb
3. fingers
How is DNA polymerase flexible but specific?
- specific: active site is used to catalyze 4 different reactions (addition of the 4 dNTPs)
- flexible: geometry of A:T, T:A, G:C, and C:G base pairs are similar, but very different from any mismatched base pairs. So what prevents the wrong base pair from being added? When a correct base pair is formed between the template and incoming dNTP, the 3’OH of the primer is positioned properly to attack the incoming alpha-phosphate (forming a new phosphodiester bond). When an incorrect base pair forms, the alpha phosphate is displaced from the site of catalysis, strongly reducing the rate of catalysis up to 10,000 fold. “kinetic proofreading”
How does DNA polymerase distinguish between rNTPs (uracil) and dNTPs (thymine)?
The dNTP’s alpha phosphate is in the catalytic center, ready for catalysis. The rNTP’s alpha phosphate is displaced from the catalytic center.
What substrates are used for DNA replication?
- dNTPs: dGTP, dCTP, dATP, dTTP
2. RNA Primers: a short sequence of nucleotides that is bp to the template with a 3’OH end of growing DNA
How does DNA polymerase distinguish between rNTPs (uracil) and dNTPs (thymine)?
The dNTP’s alpha phosphate is in the catalytic center, ready for catalysis. The rNTP’s alpha phosphate is displaced from the catalytic center.
How are two metal ions (Mg2+) in the active site of DNA Polymerase, critical for catalysis?
- one generates a 3’O that is ready for catalysis by reducing the affinity of the 3’OH for its H
- one neutralizes the negative charges on the triphosphate and stabilizes the pyrophosphate generated from catalysis
T/F. Polymerase closes and reopens its grip with each nucleotide added.
true
The replication fork
The junction between the unreplicated DNA duplex and the newly separated template strands
- both strands of DNA are replicated at the same time and must be separated to serve as templates for DNA synthesis
- leading strand is in the same direction as the replication fork 5’-3’
- lagging strand is in the opposite direction of the replication fork 5’-3’
Leading strand synthesis
-synthesized in 5’-3’ direction which is the same direction as the direction of the replication fork movement
