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 reproductiona. Bacteriab. 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. Conservativeb. Semi-conservativec. 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. colib. 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 nitrogenb. Over time this isotope gets incorporated into DNA c. DNA containing 15N is more dense than DNA containing the normal nitrogen isotope 14Nd. 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 isotopeb. 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 14Nb. 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 14Nb. 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 replicationb. DNA polymerase αc. DNA polymerase δd. DNA polymerase ε

3. The DNA polymerases require nucleotides (dNTPs) as substrates to catalyze synthesis of new DNA

a. Contain deoxyribose b. Nitrogenous basec. Three phosphates

B. DNA Synthesis: Addition of Nucleotides to a Growing DNA Strand

1. For all organisms, DNA synthesis occurs in the 5’ 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 and phosphates β γare lost

2. Any one of four nucleotides can be used for addition onto the growing DNA chain

a. dATPb. dTTPc. dGTPd. 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 replicationb. Rolling circle replication

5. For all methods, whether prokaryotic or eukaryotic several basic principles exist

a. DNA replication occurs in the 5’ 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’ 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’ 3’ direction in a continuous manner

b. One strand is synthesized in the 5’ 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. Cdc6b. 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 5’3’ direction

2. Three DNA polymerases carry out DNA replication in Eukaryotes, with each will have a different function

a. DNA polymerase αb. DNA polymerase δ

c. DNA polymerase ε

3. DNA polymerase and are the replicative polymerases that δ εfunction 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 and are the polymerases that function to δ εadd nucleotides onto a growing DNA strand

8. DNA polymerase is the primase because it functions to lay a αDNA 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’ 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 5’3’ away from the replication fork

3. The leading strand is synthesized in the 5’3’ direction towards the replication fork

4. DNA polymerase is thought to be the polymerase involved in εleading strand synthesis, but there may still be a role for DNA polymerase in leading strand synthesis as wellδ

5. To start leading strand synthesis, DNA polymerase cannot εbind single stranded DNA and start replication on its own

6. DNA polymerase needs a primer with a free 3’OH group to εstart synthesis

7. DNA polymerase (primase) will recognize the single αstranded DNA and lay down an RNA primer

8. This RNA primer will provide the necessary 3’ OH group that DNA polymerase will use to begin synthesis of the leading εstrand

9. The DNA polymerase will then bind the 3’ OH group and then εcatalyze strand synthesis in the 5’3’ 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 5’3’ 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

8. Reiji is credited for the actual description, however it was Tsuneko’s work that led to the discovery of the fragments

9. The critical DNA polymerase in lagging strand synthesis is DNA polymerase δ

10. Like DNA polymerase , DNA polymerase cannot bind or ε δstart DNA synthesis de novo (on its own)

11. DNA polymerase will lay down a primer at the site of the αreplication fork

12. As the replication fork moves away from the origin of replication, new single stranded DNA will become available and another primer will be laid down

13. The addition of each primer will result in the formation of another fragment of DNA by elongation using DNA polymerase δ

14. Each fragment that is produced is considered an Okazaki Fragment

15. After each RNA primer is laid down a 3’ OH group is available

a. Allows DNA polymerase to bind δb. Catalyze synthesis of DNA

16. For each Okazaki Fragment, DNA polymerase synthesizes δDNA in the 5’3’ direction adding nucleotides onto the 3’ end of the growing strand

17. Addition of new nucleotides results in a phosphodiester bond between the 3’OH of the previous nucleotide and the 5’ PO4 of the newly added nucleotide

18. The nucleotide added should have the complementary nitrogenous base to that on the template strand

19. Each fragment is synthesized by the DNA polymerase until εthe point where the DNA polymerase reaches the next δfragment

H. Eukaryotic Linear DNA Synthesis: Piecing Together The Lagging Strand

1. In order to complete lagging strand synthesis, the Okazaki Fragments must be pieced together

2. The first step in piecing together the Okazaki fragments is to remove the RNA primers

3. There are two ways to remove the RNA primer

4. The first mechanism involves RNase H1 nicking the RNA primer

a. Breaks the hydrogen bonds between base pairs in the RNA primer and the DNA

b. RNase H1 will leave the last base pair before the RNA-DNA junction

5. Then, the FEN-1 endonuclease degrades the primer in the 5’3’ direction

a. Has endonuclease activityb. Has exponuclease activity

6. Then, DNA polymerase will fill in the gap between Okazaki δfragments by using the 3’ OH of the upstream fragment to start gap fill-in

a. Needs that 3’ OH to bind DNAb. 3’OH is also the place to start adding nucleotides complementary to template sequence

J. Eukaryotic Linear DNA Synthesis: Piecing Together The Lagging Strand (Second Model)

1. The second proposed model is simpler than the first

2. In the second model, as DNA polymerase is synthesizing an δOkazaki fragment, it will reach the RNA primer of the next Okazaki fragment

3. When it reaches the next fragment, it will cause displacement of the RNA downstream RNA primer, and in its place, it will add nucleotides that are complementary to the template

4. Meanwhile, the FEN-1 endonuclease will then degrade the RNA primer

5. Note, in this mechanism that the RNase H1 is not required

6. Although it is not entirely clear which of the two mechanisms is really happening in vivo, data suggests that the real mechanism is the first

K. Eukaryotic Linear DNA Synthesis: Removing Primer On The Leading Strand

1. Remember, we also used an RNA primer to start synthesis of the leading strand

2. This primer must also be removed before and DNA sequence filled in before leading strand synthesis is complete

3. To remove the RNA primer we must first look at the replication bubble

4. At the origin of replication, the DNA polymerase used in εsynthesis of the last Okazaki fragment, will reach the RNA primer for the leading strand

5. Figure 6.9: Eukaryotic Linear Synthesis: Removing Primer On The Leading Strand

L. Eukaryotic Linear DNA Synthesis: Making DNA Polymerases More Efficient By Formation Of The Sliding Clamp

1. Function of the sliding clamp is to increase DNA polymerase processivity

a. Allows the appropriate replicative DNA polymerase to add nucleotides over a longer distance

b. Torque caused by the production of the new double stranded helix would cause the appropriate replicative DNA polymerase to lose its place at the replication fork

2. In order to solve the problem of the DNA polymerase from losing its place, a complex called PCNA (proliferating cell nuclear antigen) acts as a sliding clamp

a. PCNA functions as a trimer (complex of three proteins)

b. Three identical PCNA monomers are joined in a head to tail arrangement to form a ring shaped structure

3. The DNA double helix is encircled by the PCNA trimer, which will allow for the DNA polymerase to continually relax and then regain its hold

4. Without PCNA, torque caused by the production of the new Double stranded helix would cause the polymerase to lose its place at the replication fork. PCNA allows the polymerase to relax and regain its hold

5. A protein complex called RFC places the PCNA onto the DNA

a. Acts as a “clamp loader” and consists of 5 subunits

b. In the presence of ATP, RFC opens the PCNA trimer and opens the PCNA trimer and passes the DNA into the ring and then reseals it

c. Structural analysis suggests that RFC may interact with the minor grooves in DNA

6. Allows For the Polymerization to occur on the order of thousands of nucleotides

7. Figure 6.7: Formation Of The Sliding Clamp

M. Eukaryotic Linear DNA Synthesis: Termination of DNA Synthesis

1. We’ve synthesized the leading and lagging strands, but the process must be finished

2. The mechanisms of how replication actually terminates (ends) are still largely unknown

3. Replication is thought to occur until the next fork is reached

4. Some sequences have been found and shown to be able to halt replication forks

N. Eukaryotic Linear DNA Synthesis: Replicating The Ends Of The Chromosomes (Telomeres)

1. To deal with the problem of the ends being shortened after each round of repliction due to removal of the RNA primer from the lagging strand, telomeres are created

a. At the completion of lagging strand synthesis there is a shortened 5’ strand

b. There is a 12-16 nt overhang from the 3’ strand due to the removal of that 5’ primer

c. Telomeres are known to cap the ends of linear chromosomes such that they do not remain shortened

2. First identified by Barbara McClintock (1938) working with maize, and defined by H.M. Muller in Drosophila

3. Telomeres are comprised of tandem repeats of a guanine (G) rich sequence

4. Sequence is evolutionarily conserved from yeast to ciliates to plants to mammals

5. Human telomeres contain thousands of repeats of the sequence TTAGGG, whereas tetrahymena has telomere sequence TTGGGG

O. Eukaryotic Linear DNA Synthesis: Creation of Telomeres

1. Telomeres maintain chromosomal length and seal the ends of chromosomes

2. Confer stability by keeping the chromosomes from ligating together

3. Loss of telomeres leads to end-to-end chromosome fusions, facilitates genetic recombination and triggers cell death

P. Eukaryotic Linear DNA Synthesis: Solutions To The End Replication Problem – Discovery Of The Telomerase Enzyme

1. McClintock discovered telomeres at chromosomal ends, however it was still unknown how this was the solution to the end replication problem for the lagging strand

2. Greider and Blackburn (1989) discovered how telomeres were added by working with the small single-celled Eukaryote Tetrahymena thermophila

a. Tetrahymena has many chromosomes

b. Tetrahymena will have many telomeres (40,000) (Note that there can be more than two per chromosome

c. Expected that the machinery that added telomeres would be abundant in these organisms

d. The same year telomerase activity was found in human cells

3. Greider and Blackburn discovered the enzyme terminal transferase (Telomerase) which catalyzed de novo addition of telomeric repeats to the end of chromosomes

a. Telomerase enzyme is a ribonucleoprotein (RNP) complex (The enzyme has an RNA and a protein component)

b. The first component purified was the telomerase RNA component called TERC (template for telomerase RNA compoent)

c. Each subunit of telomerase has an important functionThe RNA component functions as a template for telomere addition

d. The protein component functions as a reverse transcriptase

4. Figure 6.20: Eukaryotic Linear DNA Synthesis: Solutions To The End Replication Problem – Discovery Of The Telomerase Enzyme

Q. Eukaryotic Linear DNA Synthesis: Mechanism Of Telomere Addition

1. Surprisingly, telomerase does not extend the short 5’ (lagging strand)

2. Telomerase instead interacts with the 3’ end of the lagging strand template

a. Causes elongation of the 3’ template of the lagging strand

b. Adds one telomere repeat at at time

c. Repositioning allows for further additions of repeats (one at a time)

d. Addition occurs in the 5’ 3’ direction by the telomerase enzyme

3. Once the telomerase has added telomere to the 3’ end of the lagging strand template, synthesis of the lagging strand end can occur

a. DNA polymerase can then lay an RNA primerα

b. DNA polymerase performs the elongation stepε

R. Eukaryotic Linear DNA Synthesis: Regulation of Telomere Length

1. Although telomeres are important in maintaining appropriate chromosome length, it is also important that they do not become too long (length is regulated)

2. Three proteins play a role in maintaining the appropriate telomere length

a. POT1b. TRF1c. TRF2

3. POT1 (protection of telomeres) binds the 3’ end single stranded DNA tail while TRF1 and 2 (TTAGGG repeat binding factors) prevents telomerase access by forming a folded chromatin structure

4. It is unknown how this system functions to sense telomere length

5. It is clear that the amount of POT 1 present acts as a sensor (when enough POT1 is present) this leads to a shut down telomere addition

S. Eukaryotic Linear DNA Synthesis: Telomerase, Aging And Cancer

1. In most uni-cellular organisms, telomerase has a housekeeping function – core components are always expressed

2. Human cells that do not divide rapidly do not produce high quantities of telomerase and cannot maintain telomere length during cycles of replication – Each round of cell division for these cells, the telomeres get shorter

3. Rapidly dividing cells maintain high level of telomerase, and telomere length does not shorten

4. Telomere shortening is a proposed “molecular clock” that serves to count the number of cell divisions

5. Once the telomeres reach a certain length (shortened), proliferative arrest occurs (senescence): usually at about 20 divisions

T. Eukaryotic Linear DNA Synthesis: Telomerase, and aging- Two Experiments Show The Connection Between Telomerase And Aging

1. Experiment 1: Knockout of the telomerase RNA

a. Wild-type cells can divide well up to 450 divisions (note: mice telomeres are longer, and it takes 300 divisions, for mouse cells to reach senescence)

b. Telomerase deficient mouse cells may stop dividing as only about 6 divisions, and premature aging was observed

2. Experiment 2: The reverse transcriptase (hTERT) component of the telomerase is overexpressed in human somatic cells (Note: the reverse transcriptase component is limiting)

3. Overexpression of telomerase results in immortalization

4. Figure 6.22 Eukaryotic Linear DNA Synthesis: Telomerase, and Aging- Two Experiments Show The Connection Between Telomerase And Aging

V. Prokaryotic DNA Replication

A. Prokaryotic DNA Replication: Introduction

1.Prokaryotes commonly have two types of DNA molecules

a. Basically all bacteria have a circular chromosome containing most if not all of their genes

b. Some may contain plasmids-the plasmids are small circular DNA molecules that can contain genes conferring antibiotic resistance

2. There are two different mechanisms by which bacteria can replicate their DNA

a. Rolling Circular Replication (Plasmids)b. Bi-Directional Replication (Theta Replication-Chromosomes)c. Uni-Directional Replication

B. Prokaryotic DNA Replication: Rolling Circle Replication

1.. Rolling circle replication is used to replicate some plasmids

2. Plasmids are small circular DNA molecules that are double stranded

3. For rolling circle replication, the outer strand is considered the (+) strand and the inner strand is the (-) strand

4. To start rolling circle replication, the (+) strand is nicked (cut)

5. In rolling circle replication, the (+) strand serves as a template for the (-) strand and the (-) strand serves as a template to make the (+) strand

6. The nick in the (+) strand will create a free 3’ OH and allow for synthesis of a new (+) strand

a. Synthesis of a new (+) strand will start at the free 3’OH using the (-) strand as a template

b. Synthesis of a new (+) strand is continuous lending the name rolling circle because we go around the circle

7. Synthesis of a new (-) strand uses the (+) strand as a template

8. Synthesis of a new (-) strand occurs in a discontinuous replication

C. Prokaryotic DNA Replication: Bi-Directional Replication In Bacteria (Theta Replication) Introduction

1. Bi-direction DNA replication is also known as theta replication

2. It is called theta replication because the intermediate created during DNA synthesis look like the Greek letter theta

3. In Bi-Directional replication, two replication forks are created at an origin of replication, just as in eukaryotic replication with BOTH forks moving as the DNA is being replicated (similar to eukaryotes)

4. Replication ends as the forks meet each other at the other end of the circular chromosome (Prokaryotes only)

D. Prokaryotic DNA Replication: Evidence For Two Replication Forks In Bacteria

1. John Cairns is an English Molecular Biologist, who first uncovered evidence for two replication forks in Bacterial chromosome replication

2. John Cairns worked with replicated E. coli chromosomal DNA

3. E. coli chromosomal DNA is circular

4. He labeled replicating E. coli DNA with a radioactive nucleotide for one full round and part of a second round of replication

5. After the first round, the DNA should have two strands

a. One strand is radiolabeled (new)b. One strand is not radiolabeled (old-template)

E. Prokaryotic DNA Replication: Evidence For Bi-Directional Replication In Bacteria

1. Although there are two replication forks created, there are two possible ways that the circular Bacterial chromosome could be replicated

a. Uni-directional (DNA is replicated at only one of the forks)b. Bi-Directional (DNA is replicated at both forks)

2. Elizabeth Gyurasits and R.B. Wake showed that DNA replication in B. subtilis is bi-directional

3. Grew B. subtilis in the presence of 3H-Thymidine (weak radioactivity) for a short time

4. Then switch the B. subtilis to media with a more strongly radiolabeled nucleotide (32P)

5. Exposed the replicating DNA to autoradiography

6. Both forks picked up the strongly radiolabeled nucleotide, and must have been replicating at the point where the shift occured

F. Prokaryotic DNA Replication: Evidence For Two Replication Forks In Bacteria

1. For the next round of replication, when the DNA is unwound, one template strand will be radiolabeled and the other strand will not

2. Therefore, when replicating in the second round one newly forming double stranded DNA molecule will have one strand labeled, and the other newly forming DNA molecule will have both strands labeled.

3. The structure formed during the second round of replication looks like a theta ( )θ

4. The results from the newly forming molecule with two radiolabeled strands showed that there were two replication forks

G. Prokaryotic DNA Replication: Starting Bi-Directional Replication

1. Just like in eukaryotes, replication must occur at an origin of replication

2. Unlike in Eukaryotes, there is only going to be one origin of replication

a. The bacterial chromosome is circularb. The bacterial chromosome is significantly smaller than a eukaryotic chromosome

3. In the E. coli chromosome, the origin of replication has a specfic name

a. The origin of replication is called OriC b. OriC has four 9-mers of the sequence TTATCCACA and three 13-mer repeats

4. The first step in Prokaryotic chromosomal replication is to unwind the DNA

5. To unwind the DNA, initiator proteins will bind the OriC and unwind a small section of the DNA

6. Then a protein called DNA helicase will further break hydrogen bonds existing between base pairs the two strands to unwind the DNA (note: DNA helicase will move in the 5’ 3’ direction along the lagging strand template)

7. Once the DNA is unwound, two replication forks are created going in opposite directions

8. When the DNA is unwound, then there are single stranded templates to use for replication

H. Prokaryotic DNA Replication: Unwinding the DNA and Building A Primosome Complex At The Origin Of Replication

1. The DNA is unwound at the OriC and requires several proteins

a. DnaAb. DnaB (helicase)c. DnaC

2. The nine-mers of OriC function as a binding site for DnaA

3. Once DnaA binds, it will then help facilitate the binding of DnaB

a. DnaA helps DnaB bind the origin by stimulating the melting of the three 13-mer repeats just to the left of OriC

b. DnaB will also need the aid of DnaC to bind the OriC

c. DnaB is a helicase, and catalyzes the formation of two replication forks at an origin of replication

d. DnaB requires ATP to catalyze formation of replication forks

4. This protein complex, plus the RNA polymerase and HU protein form the primosome complex

J. Prokaryotic DNA Replication: Bi-Directional Replication and the Replicative DNA Polymerases

1. Once the DNA is unwound, replication occurs in much the same way as it does in Eukaryotes (with both leading and lagging strands)

a. Proteins mediate the process of replication although they are different than in eukaryotes

b. Nucleotides to serve as raw materials to make the DNA

c. Single stranded DNA serves as a template (The replication is semi-conservative)

2. Just like in eukaryotic replication DNA polymerases mediate the synthesis of the new DNA

a. DNA polymerase I (pol I) fills in the gaps between Okazaki Fragments

b. DNA polymerase II (pol II)

c. DNA polymerase III (pol III) synthesizes both leading and lagging strands

K. Prokaryotic DNA Replication: Bi-Directional Replication and Leading Strand Synthesis

1. As previously stated, bi-directional replication in bacteria occurs in a similar manner as eukaryotes

a. Both leading strand and lagging strand synthesis

b. Both forks are moving away from the origin of replication

2. Just like the eukaryotic DNA polymerases, DNA polymerase III (pol III) requires a free 3’ OH group to be able to bind DNA and start the synthesis

3. Therefore, an RNA primer must be laid down before pol III can then mediate synthesis on both leading and lagging strands

4. DnaG (also known as primase) lays down an RNA primer (~10-12 nt) long, and this primer will provide the free 3’OH5. Just like the eukaryotes, the leading strand is synthesized in a continuous manner

6. The template for the leading strand is the strand that goes 5’3’ away from the replication fork

7. The leading strand is synthesized in the 5’3’ direction towards the replication fork

8. In prokaryotes, DNA polymerase III will bind the DNA at the free 3’OH and add nucleotides with nitrogenous bases that are complementary to the sequence in the template

L. Prokaryotic DNA Replication: Bi-Directional Replication and Lagging Strand Synthesis

1. Just like in eukaryotic replication, lagging strand replication in prokaryotes is also discontinuous

2. In prokaryotic replication, lagging strand synthesis is done by producing Okazaki fragments

3. Since lagging strand synthesis is discontinuous, a primers must be regularly laid down

4. In order to do this, at the beginning of replication DnaG binds DNA helicase, and moves along the lagging strand template with DNA helicase, laying down RNA primers as the DNA becomes single stranded

5. pol III will then bind the DNA at the free 3’OH and add nucleotide to synthesize new DNA

6. Now that we’ve synthesized the Okazaki fragments, we must remove the RNA primers, and fill in the sequence

7. On the lagging strand, DNA polymerase III will synthesize DNA until it reaches the next Okazaki fragment

8. DNA polymerase I (pol I) has 5’3’ exonuclease activity and polymerase activity

9. DNA polymerase I will then remove the RNA primer and fill in the sequence using the final 3’OH group of the upstream Okazaki fragment

10. After DNA polymerase I has replaced the last nucleotide, only a nick remains

11. This nick is due to the fact that the 3’ OH last nucleotide added by DNA polymerase I has not been joined to the 5’ PO4 of the first DNA nucleotide in the next fragment

12. To join the 3’ OH and the 5’ PO4, DNA ligase is used

M. Prokaryotic DNA Synthesis: Termination of Bi-Directional DNA Synthesis

1. Unlike Eukaryotic DNA replication, the mechanism of how DNA replication is terminated in prokaryotes is much better understood

2. As DNA synthesis occurs, the two replication forks will move around the circular chromosome

3. When these replication forks meet, replication then terminates, and we have two new DNA molecules

4. Termination of replication occurs when the two forks meet each other on the opposite side of the chromosome from the OriC

5. Termination occurs at termination sites (Ter A-F)

6. Each replication fork as it moves around the circular chromosome moves a different speeds

7. The termination sites are bound by a protein Tus, which plays a role in halting the replication forks

a. Leading strand synthesis arrests at the point of contact with Tus

b. The final lagging strand RNA primer sites are 50-70 nt away from Tus

8. Based on the speed of how the replication forks are moving three possible scenarios for termination exist

9. Scenario 1: If the clockwise moving fork is moving faster and reaches Ter F,B,C, it will stop

a. Somehow the clockwise moving fork will get through the TerA, TerD and TerE

b. The clockwise fork will wait for the counter-clockwise fork to meet it

10. Scenario 2: If the counter-clockwise is moving faster and then reaches Ter A, D, E, it will stop

a. Somehow the counter-clockwise moving fork will get through the Ter F, TerB, TerC sites

b. The counter-clockwise fork will wait for the clockwise moving fork

c. Scenario 3: Replication forks are moving at close to equivalent speeds and can meet between the Ter A,D,E cluster and the Ter F,B,C cluster

N. Prokaryotic DNA Replication: Resolving The Two DNA Molecules at the end of Bi-Directional DNA replication

1. Since prokaryotic chromosomes are circular (unlike in eukaryotes), near the end of replication, the new chromosomes are entwined as two interlocking rings (catenae)

2. Chromosomes need to be unlinked through a process called decatenation before they move to the two new daughter cells

3. Denaturation of the remaining double helix will leave some single stranded DNA where the replication forks meet

4. DNA repair synthesis will fill in the gaps where the DNA is single stranded