Chapter 6: DNA Replication and Telomere Maintenance
I. Introduction
A. Introduction: Reasons For DNA Replication
1. For life to be sustained, cells must divide and give rise to more cells
2. Each new cell must have a full copy of the genome
3. For some cells, division means reproduction
a. Bacteria
b. Unicellular Fungi
4. Development
5. Wound Repair
B. Introduction: Defects In DNA Replication Have Important Consequences
1. DNA replication is critical to all life on earth, including all simple unicellular organisms like bacteria as well as complex unicellular organisms like humans
a. Important for cell division as each new cell needs a full copy of the genetic information to function properly
b. Important for reproduction (both mitosis and meiosis)
2. Severe defects in DNA replication will almost always lead to a loss of survival
3. Other defects that affect DNA synthesis and repair result in genetic disease
4. One example of a genetic disease linked to defects in DNA synthesis and repair is Xeroderma Pigmentosum
C. Introduction: Why Replication?
1. It is important before cell division that the whole genome (all DNA) is replicated
2. Allows for each new daughter cell to have a complete copy of all DNA sequences
3. In order to do this, replication requires decisions of when to synthesize the DNA, such that it is done in a complete and accurate way before the cell starts dividing
II. Mechanisms of DNA Replication
A. Mechanisms of DNA Replication: Introduction
1. When replicating DNA we are taking one double stranded DNA molecule and making an exact copy of it
2. Based on the double stranded structure there are three mechanisms by which DNA can be replicated
a. Conservative
b. Semi-conservative
c. Dispersive
3. In conservative replication, there are two products
a. Original double stranded molecule (contains the original two strands of DNA)
b. The new double stranded molecule of DNA (contains two newly produced strands)
4. In semiconservative replication, each double stranded DNA product will consist of 1 newly produced DNA strand and 1 original strand
5. In dispersive replication, some parts of the original helix are conserved (original DNA) and some parts are newly synthesized (new DNA)
6. Supplemental Figure: Mechanisms of DNA Replication: Introduction
B. Mechanisms of DNA Replication: The Meselson-Stahl Experiment
1. In order to determine which of the three mechanisms of DNA replication were correct, Matthew Meselson, and Franklin Stahl designed an elegant experiment (1958)
a. Studied DNA replication in E. coli
b. Took advantage of the fact that DNA is nitrogen rich (nitrogenous bases)
2. Meselson and Stahl grew E. coli in medium containing 15N for several generations
a. Heavy isotope of nitrogen
b. Over time this isotope gets incorporated into DNA
c. DNA containing 15N is more dense than DNA containing the normal nitrogen isotope 14N
d. After this treatment, the E. coli had DNA with both strands containing 15N
3. Next, they shifted the E. coli to media containing 14N
a. Normal nitrogen isotope
b. Did this for only 1 round of replication
B. Mechanisms of DNA Replication: The Meselson-Stahl Experiment
1. Isolated DNA from cells and did density-gradient centrifugation using a CsCl gradient
a. At the bottom, the concentration of CsCl is high (the solution is more dense) and at the top, the concentration of CsCl is low (less dense)
b. Layered their DNA sample on the top of the gradient and centrifuged the sample
2. During centrifugation, the DNA will be pulled towards the bottom of the tube by the centrifugal force
3. The DNA will stop moving toward the bottom when it reaches a concentration of CsCl in the tube of equal density to the DNA
4. DNA Molecules can be observed within the gradient with UV light at A260
5. If you have conservative replication then you should have a DNA molecules at the top of the gradient and a double stranded DNA molecule at the bottom
a. One double stranded DNA molecule contains only 14N
b. One double stranded DNA molecule contains only 15N
5. If you have semi-conservative replication, then you should have DNA molecules in the center of the gradient
a. One strand of each molecule will contain 14N
b. One strand of each molecule will contain 15N
6. If dispersive replication, then DNA molecules would be located throughout the gradient
7. Meselson and Stahl saw their DNA run towards the center of the gradient indicating semi-concervative replication
8. Supplemental Figure: Mechanisms of DNA Replication: The Meselson-Stahl Experiment
III. DNA Synthesis
A. DNA Synthesis: Introduction
1. In semi-conservative replication, the existing DNA Molecule will serve as a template
a. Template: Original molecule which serves as a guide to make a new molecule
b. Each strand will serve as a template
c. The new strand will be complementary to the template
2. DNA synthesis does not happen De Novo (spontaneously), but requires specifc enzymes called DNA polymerases
a. Multiple DNA polymerases carry out replication
b. DNA polymerase α
c. DNA polymerase δ
d. DNA polymerase ε
3. The DNA polymerases require nucleotides (dNTPs) as substrates to catalyze synthesis of new DNA
a. Contain deoxyribose
b. Nitrogenous base
c. Three phosphates
B. DNA Synthesis: Addition of Nucleotides to a Growing DNA Strand
1. For all organisms, DNA synthesis occurs in the 5’ à 3’ direction
a. Nucleotides (dNTP) are added onto the 3’ end of the growing strand with new phosphodiester bonds being formed
b. In the condensation reaction, the β and γ phosphates are lost
2. Any one of four nucleotides can be used for addition onto the growing DNA chain
a. dATP
b. dTTP
c. dGTP
d. dCTP
3. The choice of nucleotide to add to the growing strand is determined by complementary base pairing with the template strand (which is antiparallel)
4. This is why DNA replication is semi-conservative
a. The template strand is from the original double stranded DNA molecule
b. We are using the template to produce the new strand
C. DNA Synthesis: Prokaryotic vs. Eukaryotic
1. Mechanisms of DNA replication are slightly different in prokaryotes as compared to eukaryotes
2. The difference in replication mechanisms comes from the fact that prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear
3. For eukaryotes, the DNA undergoes linear replication
4. For prokaryotes, two methods of DNA replication exist
a. Theta replication
b. Rolling circle replication
5. For all methods, whether prokaryotic or eukaryotic several basic principles exist
a. DNA replication occurs in the 5’ à 3’ direction, using a template that is antiparallel
b. DNA replication begins at sites known as origins of replication
6. For all methods, whether prokaryotic or eukaryotic several basic principles exist
a. DNA replication occurs in the 5’ à 3’ direction, using a template that is antiparallel
b. DNA replication begins at sites known as origins of replication
7. DNA replication for eukaryotes, prokaryotes as well as most DNA viruses is semi-discontinuous
a. One strand is synthesized in the 5’ à 3’ direction in a continuous manner
b. One strand is synthesized in the 5’ à 3’ direction in a discontinuous manner
IV. Eukaryotic Linear DNA Synthesis
A. Eukaryotic Linear DNA Synthesis: Origins of Replication
1. In Eukaryotes, the chromosomes are linear and quite long
2. The first thing to think about when replicating the chromosomes is where to start
3. The starting point for DNA replication is at sites that are called origins of replication
a. At origins of replication, the double stranded DNA helix is unwound
b. Unwinding creates regions that are no longer double stranded, but single stranded
c. Each single strand will serve as a template for DNA replication (DNA replication is semi-conservative)
4. On each human chromosome, it is estimated that there are between 10,000 and 100,000 origins of replication
5. Human origins of replication lack a consensus sequence, but are thought to be A-T rich (Have many A-T base pairs)
B. Eukaryotic Linear DNA Synthesis: Unwinding the DNA At Origins of Replication
1. Now that we have origins of replication, how is it that the DNA is unwound?
2. Before the DNA is unwound at origins, the histones are first removed by a yet to be determined process-This loosens the DNA
3. The first step in unwinding of the DNA is the recognition of the origin of replication by the Origin Recognition Complex (ORC)
4. The ORC will bind to each origin that will be activiated in replication
5. The ORC is an ATP-regulated DNA binding complex composed of 6 subunits (ORC 1-6)
6. Once the ORC binds the Origin of Replication, it will recruit two more proteins
a. Cdc6
b. Cdt1
7. The combined ORC, Cdc6 and Cdt1 complex is considered the Pre-replication complex
8. The Pre-replication complex will recruit the Mcm2-7 complex (Mcm stands for mini-chromosome)
9. Once Mcm2-7 complex binds, Cdc6 and Cdt1 dissociate from the DNA
10. The Mcm2-7 has helicase activity
a. Helicases are enzymes that can act to unwind DNA
b. Once Mcm2-7 acts on the DNA, it is unwound and single stranded in the region where the origin is
11. Once the DNA is single stranded, the RPA protein will bind the single stranded DNA to ensure it remains single stranded
12. When the Mcm2-7 complex unwinds the DNA, a replication bubble forms with 2 replication forks
a. The bubble is the open single-stranded DNA
b. Each fork is the junction where single stranded DNA meets double stranded DNA
c. The replication fork is where the DNA will be unwound as DNA replication proceeds
13. Once the DNA is unwound, the Mcm2-7 complex will stay associated with the DNA
14. Mcm2-7 will move away from the origin as replication proceeds, creating new areas of single stranded DNA
15. You can think about Mcm2-7 complex moving the replication forks away from the origin of replication
16. At each origin of replication, there are two forks created that move in opposite directions which actually create the replication bubble
C. Eukaryotic Linear DNA Synthesis: DNA Polymerases
1. Once the DNA is single stranded, DNA replication can be carried out by the enzymes known as DNA polymerases
a. There are three different DNA polymerases that are involved in eukaryotic replication
b. Each of the DNA polymerases can catalyze formation of the new strands of DNA only in the 5’à3’ direction
2. Three DNA polymerases carry out DNA replication in Eukaryotes, with each will have a different function
a. DNA polymerase α
b. DNA polymerase δ
c. DNA polymerase ε
3. DNA polymerase δ and ε are the replicative polymerases that function to add nucleotides onto a growing DNA strand
4. DNA Polymerases are high-fidelity enzymes: they replicate the DNA without many errors
5. Replicative DNA polymerases are not perfect with mutation rates ranging from 10-4 to 10-5 per base pair (an error once every 10,000-100,000 base pairs)
6. Replicating DNA polymerases contain a proofreading exonuclease that can excise 90-99% of misincorporated nucleotides
7. DNA polymerase δ and ε are the polymerases that function to add nucleotides onto a growing DNA strand
8. DNA polymerase α is the primase because it functions to lay a DNA primer
D. Eukaryotic Linear DNA Synthesis: Problems Associated With Replication Fork Generation
1. Movement of the replication fork machinery causes supercoiling of the DNA ahead of the fork
2. Supercoiling of the DNA causes torsional stress which could block replication fork movement
3. Supercoiling ahead of the fork is resolved by topoisomerase I and II (enzymes that function to unwind DNA)
E. Eukaryotic Linear Replication: Introduction To The Leading and Lagging Strands
1. If you look at a single replication fork, if DNA polymerase synthesizes the new strands in the 5’ à 3’ direction, one strand will be synthesized away from the replication fork and one towards the replication
2. The strand that gets synthesized going towards the replication fork is the leading strand
3. The strand that gets synthesized going away from the replication fork is called the lagging strand
4. The leading and lagging strands get synthesized in a very different manner
F. Eukaryotic Linear Replication: Leading Strand Synthesis
1. The leading strand is the easiest strand to synthesize namely because it occurs continuously
2. The template for the leading strand is the strand that goes 5’à3’ away from the replication fork
3. The leading strand is synthesized in the 5’à3’ direction towards the replication fork
4. DNA polymerase ε is thought to be the polymerase involved in leading strand synthesis, but there may still be a role for DNA polymerase δ in leading strand synthesis as well
5. To start leading strand synthesis, DNA polymerase ε cannot bind single stranded DNA and start replication on its own
6. DNA polymerase ε needs a primer with a free 3’OH group to start synthesis
7. DNA polymerase α (primase) will recognize the single stranded DNA and lay down an RNA primer
8. This RNA primer will provide the necessary 3’ OH group that DNA polymerase ε will use to begin synthesis of the leading strand
9. The DNA polymerase ε will then bind the 3’ OH group and then catalyze strand synthesis in the 5’à3’ direction towards the replication fork
10. As the replication fork moves away from the origin of replication, the leading strand will continue to be synthesized, essentially, into the replication fork
11. As the replication fork keeps moving, DNA can be synthesized continuously
G. Eukaryotic Linear DNA Synthesis: Lagging Strand Synthesis
1. Synthesis of the lagging strand is more difficult than the leading strand
2. This is because the lagging strand is synthesized in the 5’à3’ direction away from the replication fork
3. This poses a problem because as the replication fork moves, where do you start the synthesis of the strand?
4. In order to synthesize the lagging strand, this can’t be done continuously, it must be done in a non-continuous manner
5. The lagging strand is synthesized in fragments that are then ligated (linked) together
6. The fragments that are put down in lagging strand synthesis are known as Okazaki fragments
7. The Okazaki fragments are named after Reiji and Tsuneko Okazaki