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1.5: DNA Replication - Introduction to Prokaryotic replication - Biology

1.5: DNA Replication - Introduction to Prokaryotic replication - Biology


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1953

  • The DNA model of Watson and Crick suggested how genetic information might be replicated: either strand of the duplex can be used as a template to replicate the sequence information.
  • But, was the replication conservative (i.e. the original parental strands remain together after replication) or semi-conservative(one parental strand pairs with one newly synthesized strand)?

The answer for prokaryotic organisms (i.e. bacteria) came from the 1958 experiment of Meselson and Stahl.

1958

The nitrogen in ammonium salts in culture broth is incorporated into DNA bases. The most common isotope of nitrogen is 14N. However, 15N ammonium salts (a heavier isotope) can also be obtained.

  • DNA from E. coli cells grown with 15N ammonium salts will have a higher density than DNA grown in "normal" (14N) ammonium salts.
  • Such DNA will migrate differently on cesium chloride (CsCl2) equilibrium density gradient centrifugation.
  • The more dense DNA will migrate as a lower band (on this type of centrifugation the characteristic migration position is a function of density, and is independent of DNA length).

Figure 1.5.1: Density of 15N and 14N DNA

Meselson and Stahl reasoned that if they grew E. coli in 15N salts then switched media to 14N salts for additional rounds of replication, the mode of replication could be deduced from the density of the DNA.

Figure 1.5.2: Meselson and Stahl experiment

  • After switching to the 14N media and allowing the cells to go through a round of replication a single band of intermediate density was observed (i.e. between 14N and 15N control DNA samples).
  • After a second round of replication in 14N media two bands were present in approximately equimolar amounts; one was intermediate in density and the other migrated as purely 14N labeled DNA.

The results were consistent with a semi-conservative mode of replication for DNA. Evidence of semi-conservative replication of DNA has since been obtained with both plant and animal DNA.

DNA Replication in E. coli

E. coli DNA polymerase characteristics:

  • Polymerase will only elongate an existing polynucleotide. It cannot initiate polynucleotide formation:

Figure 1.5.3: DNA Polymerase activity

  • Polymerase will catalyze polymerization of nucleotides only in one direction (5'­>3') via a phosphodiester bond between a 3' hydroxyl and 5' phosphate group.
  • DNA polymerase is unable to unwind duplex DNA to separate the two strands which need to be copied

E. coli genome is circular duplex DNA of approximately 4 x 106 base pairs (i.e. 4 Mb)

  • The genome has a single origin of replication.
  • DNA duplication in E. coli begins at a specific site in the DNA called "oriC".

OriC is a region of DNA approximately 240 nucleotides long.

  • It contains repetitive 9-base pair and 13-base pair sequences (known as the '9-mer' and '13-mer' regions).
  • These sequences are AT rich regions, which melt at lower temperatures than DNA containing GC pairs.
  • These regions are postulated to help melt the DNA duplex in the oriC region for initiation of DNA replication.


Figure 1.5.4: oriC region

The dnaA gene product: (dnaA protein)

  • Strains of E. coli with mutations in the dnaA gene were able to grow at 30 °C, but not at 39-42 °C.
  • However, if DNA synthesis was begun at 30 °C, and then the temperature was shifted to 42 °C, DNA synthesis continued until the genome was replicated (and the cell divided), but no new initiationof DNA synthesis was possible.

Conclusion: Somehow the product of the dnaA gene (i.e. the dnaA protein) is required for initiation of DNA synthesis.

Studies of purified dnaA protein:

  • dnaA protein binds to the '9-mer' region in oriC and forming a multimeric complex with 10-20 protein subunits (i.e. at a single oriC region there will be bound 10-20 dnaA protein molecules).
  • Binding requires ATP.
  • Further addition of ATP was observed to result in a melting and opening up of the DNA duplex in the oriC region. This was determined by addition of S1 nuclease (like mung bean, but will also cut DNA at the site of an internal nick), which resulted in cleavage of DNA at the site of oriC.

The dnaB gene product: (dnaB protein)

The protein encoded by the dnaB gene appears to be essential for DNA replication. The dnaB protein has been identified as ahelicase. A helicase moves along a DNA strand opening up the duplex to melt and separate the DNA strands.

  • dnaB protein binds to the single stranded DNA in the general region of the oriC DNA segment.
  • Binding requires ATP as well as the dnaC gene product (the dnaC protein).
  • After helicase/dnaC binds to the DNA, the dnaC protein is released.
  • Two helicases bind at the oriC region, one helicase on each strand of the DNA.

This stage represents the prepriming complex:

Figure 1.5.5: Prepriming complex

Separated strands in the oriC region are prevented from reannealing by the binding of single-stranded binding protein (ssb protein).

The dnaG gene protein:

The dnaG gene protein is called primase.

  • Primase catalyzes synthesis of short RNA molecules that function as primers for DNA synthesis by E. coli DNA polymerase III(pol III).
  • Primase binds to dnaB protein at oriC and forms a primosome.
  • The primase within the primosome complex provides RNA primers for synthesis of both strands of duplex DNA.
  • Primase lays down tracks of pppAC(N)7-10 (RNA).

Figure 1.5.6: Primase activity

  • After synthesis of the 9-12 mer RNA primer, DNA Pol III holoenzyme enters the replication fork and is able to utilize the RNA as a primer for DNA synthesis.
  • As the replication fork opens up, the leading strand synthesis can continue, but a gap develops in the lagging strand:


Figure 1.5.7: Lagging Strand

DNA Pol III is a large multicomplex enzyme (holoenzyme) which is somewhat dimeric in nature (there are two polymerase active sites). The two active polymerase sites in Pol III could actually function to synthesize both nacent strands at the fork. However, the synthesis of the lagging template strand would be in the opposited direction to the movement of the Pol III complex:

Figure 1.5.8: Pol III movement

Primase can bind to the Pol III complex, but the arrangement of the DNA strand as it passes through the Pol III/primase complex is quite unique. It forms a loop structure such that primase and the Pol III active site can accomplish discontinuous synthesis of the lagging template strand even though the general direction of the Pol III complex is opposite to the require direction of DNA synthesis:

Figure 1.5.9: Loop for Pol III activity

After primase makes another primer on the lagging template, the adjacent Pol III active site can extend the primer (incorporating dNTP's) by utilizing the same loop structure and feeding the template through in the direction shown.

Figure 1.5.10: Synthesis of lagging strand

The lagging strand loop cannot be fed through the Pol III complex forever, and after a nascent DNA strand is synthesized the loop is released and a new one is formed using the opened template DNA further up the fork:

Figure 1.5.11: Continuation of lagging strand synthesis

As synthesis continues:

  • there will be a single continuous DNA strand on the leading strand
  • there will be a series of short fragments on the lagging strand, containing both RNA and DNA, called Okazaki fragments:

Figure 1.5.12: Okazaki Fragments

How are these RNA/DNA fragments converted into one long continuous DNA strand? The RNA could be removed by a polymerase which has 5'->3' exonuclease activity, however, Pol III lacks this activity.

  • DNA Pol I does have 5'->3' exonuclease activity
  • it can extend the DNA synthesis via nick-translation.
  • The nick-translation activity restults in degradation of the RNA primers.
  • The end result is a series of "nicks" in the lagging strand, now 100% DNA:

Figure 1.5.13: Nicks in lagging strand

  • DNA Pol I leaves and DNA ligase then joins these discontinuous DNA fragments to form a continuous DNA duplex on the lagging strand.

Summary of steps in E. coli DNA Synthesis

  1. dnaA protein melts duplex in oriC region.
  2. dnaB (helicase), along with dnaC and ATP binds to replication fork (dnaC protein exits).1 (Pre-priming complex)
  3. Single strand binding protein (ssb protein) binds to separated strands of DNA and prevents reannealing.
  4. Primase complexes with helicase, creates RNA primers (pppAC(N)7-10) on the strands of the open duplex2 (Primase+helicase constitute the Primosome).
  5. After making the RNA primers, DNA pol III holoenzyme comes in and extends the RNA primer (laying down dNTP's) on the leading strand.
  6. As the replication fork opens up (via helicase + ATP action) leading strand synthesis is an uninterrupted process, the lagging strand experiences a gap.
  7. The gap region of the lagging strand can wind around one active site unit of the Pol III complex, and bound Primase initiates an RNA primer in the gap region3.
  8. On the lagging strand, Pol III extends the RNA primer with dNTP's as the lagging template strand is looped through the Pol III complex
  9. After synthesis of a nascent fragment the lagging strand loop is released and the single strand region further up near the replication fork is subsequently looped through the Pol III complex.
  10. Steps 7-9 are repeated.
  11. Meanwhile, Pol I removes the RNA primer regions of the Okazaki fragments via 5' to 3' exonuclease activity ( nick translation
  12. Pol I exits and ligase joints the DNA fragments (on lagging strand).

Notes From Above

  1. Polymerases are unable to open up duplex DNA, thus the requirement for helicase
  2. Polymerases cannot replicate a DNA template in the absence of a primer (either DNA or RNA).
  3. Polymerases extend a polynucleotide in the 5' to 3' direction only. Gaps at the 5' end must be filled by "upstream" discontinuous synthesis.
Properties of E. coli polymerases (Pol I, II and III)

DNA Pol I

DNA Pol II

DNA Pol III

5'->3' Polymerase activity

*

*

*

3'->5' Exonuclease activity(proof reading)

*

*

*

5'->3' Exonuclease activity(nick translation)

*

Synthesis from:

Duplex DNA

Primed single strand

*

Primed single strand plus ssb protein

*

*

Chain elongation rate(in vitro) bp/min

600

?

30,000

Molecules/cell

400

?

10-20

Mutation Lethal?

*

*

Pol Functions:

  • Pol I: gap filling during DNA synthesis and repair, removal of RNA primers
  • Pol II: involved in DNA synthesis of damaged templates
  • Pol III: functional polymerase at the replication fork

Pol III Structure and function

  • A "holoenzyme" complex of 10 different polypeptides
  • resultant molecular weight is greater than 600 KDa (i.e. it is a large complex).
  • It is structurally an asymmetric dimer - it contains two copies of most of the polypeptides which comprise it, including two catalytic sites for nucleotide addition (i.e. polymerization).

The various protein subunits have a variety of functions:

  1. Subunits for polymerase activity: a, e, subunits
  2. Subunits to dimerize the core polymerase (t)
  3. Subunits to increase processivity (i.e. to increase the ability to synthesize long stretches w/o releasing from the DNA template): b subunits
  4. Subunits to bind b to DNA-primer substrate: (g, d, d', c, )

Termination of DNA replication

  • Specific termination sites of DNA replication exist in E. coli.
  • Termination involves the binding of the tus gene product (tus protein).
  • This protein may act to prevent helicase from unwinding DNA (will therefore halt pol III and pol I action).
  • DNA replication produces two interlocking rings which must be separated.
  • This is accomplished via the enzyme topoisomerase.

ColE1 Plasmid

E. coli can contain a small extrachromosomal element called the ColE1 plasmid. This plasmid has the following general features:

  • 6.4 Kb circular duplex DNA
  • Autonomously replicating
  • 10-15 copies per cell (i.e. per E. coli chromosome)

Although it is autonomously replicating, it does not contain an oriC type of sequence for initiation of replication, and does not undergo the same steps in replication.

  • The plasmid produces (among other things) two RNA oligonucleotides (RNA I and RNA II)
  • RNA II has complementarity to the ColE1 origin of replication, which contains an AT rich sequence
  • The bound RNA II molecule can serve as a primer for polIII
  • Since RNA II binds on one strand only, the replication of the ColE1 plasmid is unidirectional
  • RNA I has complementarity with RNAII, and such a duplex RNA cannot serve as a replication primer, thus control of the plasmid copy number is achieved by the interaction between RNA I and RNA II.

DNA Replication

DNA replicates by a semi-conservative method in which each of the two parental DNA strands act as a template for new DNA to be synthesized. After replication, each DNA has one parental or “old” strand, and one daughter or “new” strand.

Replication in eukaryotes starts at multiple origins of replication, while replication in prokaryotes starts from a single origin of replication. The DNA is opened with enzymes, resulting in the formation of the replication fork. Primase synthesizes an RNA primer to initiate synthesis by DNA polymerase, which can add nucleotides in only one direction. One strand is synthesized continuously in the direction of the replication fork this is called the leading strand. The other strand is synthesized in a direction away from the replication fork, in short stretches of DNA known as Okazaki fragments. This strand is known as the lagging strand. Once replication is completed, the RNA primers are replaced by DNA nucleotides and the DNA is sealed with DNA ligase.

The ends of eukaryotic chromosomes pose a problem, as polymerase is unable to extend them without a primer. Telomerase, an enzyme with an inbuilt RNA template, extends the ends by copying the RNA template and extending one end of the chromosome. DNA polymerase can then extend the DNA using the primer. In this way, the ends of the chromosomes are protected. Cells have mechanisms for repairing DNA when it becomes damaged or errors are made in replication. These mechanisms include mismatch repair to replace nucleotides that are paired with a non-complementary base and nucleotide excision repair, which removes bases that are damaged such as thymine dimers.


14.4 DNA Replication in Prokaryotes

By the end of this section, you will be able to do the following:

  • Explain the process of DNA replication in prokaryotes
  • Discuss the role of different enzymes and proteins in supporting this process

DNA replication has been well studied in prokaryotes primarily because of the small size of the genome and because of the large variety of mutants that are available. E. coli has 4.6 million base pairs in a single circular chromosome and all of it gets replicated in approximately 42 minutes, starting from a single site along the chromosome and proceeding around the circle in both directions. This means that approximately 1000 nucleotides are added per second. Thus, the process is quite rapid and occurs without many mistakes.

DNA replication employs a large number of structural proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, also known as DNA pol, which adds nucleotides one-by-one to the growing DNA chain that is complementary to the template strand. The addition of nucleotides requires energy this energy is obtained from the nucleoside triphosphates ATP, GTP, TTP and CTP. Like ATP, the other NTPs (nucleoside triphosphates) are high-energy molecules that can serve both as the source of DNA nucleotides and the source of energy to drive the polymerization. When the bond between the phosphates is “broken,” the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain. In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis DNA pol I is an important accessory enzyme in DNA replication, and along with DNA pol II, is primarily required for repair.

How does the replication machinery know where to begin? It turns out that there are specific nucleotide sequences called origins of replication where replication begins. In E. coli, which has a single origin of replication on its one chromosome (as do most prokaryotes), this origin of replication is approximately 245 base pairs long and is rich in AT sequences. The origin of replication is recognized by certain proteins that bind to this site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication and these get extended bi-directionally as replication proceeds. Single-strand binding proteins coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix.

DNA polymerase has two important restrictions: it is able to add nucleotides only in the 5' to 3' direction (a new DNA strand can be only extended in this direction). It also requires a free 3'-OH group to which it can add nucleotides by forming a phosphodiester bond between the 3'-OH end and the 5' phosphate of the next nucleotide. This essentially means that it cannot add nucleotides if a free 3'-OH group is not available. Then how does it add the first nucleotide? The problem is solved with the help of a primer that provides the free 3'-OH end. Another enzyme, RNA primase , synthesizes an RNA segment that is about five to ten nucleotides long and complementary to the template DNA. Because this sequence primes the DNA synthesis, it is appropriately called the primer . DNA polymerase can now extend this RNA primer, adding nucleotides one-by-one that are complementary to the template strand (Figure 14.14).

Visual Connection

Question: You isolate a cell strain in which the joining of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?

The replication fork moves at the rate of 1000 nucleotides per second. Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up it does so by causing temporary nicks in the DNA helix and then resealing it. Because DNA polymerase can only extend in the 5' to 3' direction, and because the DNA double helix is antiparallel, there is a slight problem at the replication fork. The two template DNA strands have opposing orientations: one strand is in the 5' to 3' direction and the other is oriented in the 3' to 5' direction. Only one new DNA strand, the one that is complementary to the 3' to 5' parental DNA strand, can be synthesized continuously towards the replication fork. This continuously synthesized strand is known as the leading strand . The other strand, complementary to the 5' to 3' parental DNA, is extended away from the replication fork, in small fragments known as Okazaki fragments , each requiring a primer to start the synthesis. New primer segments are laid down in the direction of the replication fork, but each pointing away from it. (Okazaki fragments are named after the Japanese scientist who first discovered them. The strand with the Okazaki fragments is known as the lagging strand .)

The leading strand can be extended from a single primer, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3' to 5', and that of the leading strand 5' to 3'. A protein called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-shaped protein that binds to the DNA and holds the polymerase in place. As synthesis proceeds, the RNA primers are replaced by DNA. The primers are removed by the exonuclease activity of DNA pol I, which uses DNA behind the RNA as its own primer and fills in the gaps left by removal of the RNA nucleotides by the addition of DNA nucleotides. The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase , which catalyzes the formation of phosphodiester linkages between the 3'-OH end of one nucleotide and the 5' phosphate end of the other fragment.

Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division.

The process of DNA replication can be summarized as follows:

  1. DNA unwinds at the origin of replication.
  2. Helicase opens up the DNA-forming replication forks these are extended bidirectionally.
  3. Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
  4. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
  5. Primase synthesizes RNA primers complementary to the DNA strand.
  6. DNA polymerase III starts adding nucleotides to the 3'-OH end of the primer.
  7. Elongation of both the lagging and the leading strand continues.
  8. RNA primers are removed by exonuclease activity.
  9. Gaps are filled by DNA pol I by adding dNTPs.
  10. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.


Table 14.1 summarizes the enzymes involved in prokaryotic DNA replication and the functions of each.


Replication in prokaryotes starts from a sequence found on the chromosome called the origin of replication—the point at which the DNA opens up. Helicase opens up the DNA double helix, resulting in the formation of the replication fork. Single-strand binding proteins bind to the single-stranded DNA near the replication fork to keep the fork open. Primase synthesizes an RNA primer to initiate synthesis by DNA polymerase, which can add nucleotides only in the 5' to 3' direction. One strand is synthesized continuously in the direction of the replication fork this is called the leading strand. The other strand is synthesized in a direction away from the replication fork, in short stretches of DNA known as Okazaki fragments. This strand is known as the lagging strand. Once replication is completed, the RNA primers are replaced by DNA nucleotides and the DNA is sealed with DNA ligase, which creates phosphodiester bonds between the 3'-OH of one end and the 5' phosphate of the other strand.

Figure You isolate a cell strain in which the joining together of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?

Figure DNA ligase, as this enzyme joins together Okazaki fragments.


8. Understand the use of inhibitors of DNA synthesis as drugs, e.g. in cancer.

● Inhibition of DNA synthesis ○ For killing infections and treating cancer ○ Nucleoside analog: has OH at the 3’ instead of the phosphate groups For preventing creation of a phosphodiester bond ■ High affinity for reverse transcriptase ■ Reverse transcriptase: ● RNA-dependent DNA pol. (5’ → 3’) ● Ribonuclease H (5’ → 3’ exonuclease) ● DNA dependent DNA pol. (5’ → 3’)

○ Remove the normal OH group and replace it with certain groups to create these antiviral drugs: ■ AZT: replace OH with Azide (N 3 - ) ● High affinity for reverse transcriptase (RNA → DNA) ● 3 activities of reverse transcriptase : ○ 1. RNA-dependent DNA polymerase (5’ → 3’) (copies RNA template) ○ 2. Ribonuclease H (5’ → 3’ exonuclease) (degradation of RNA) ○ 3. DNA-dependent DNA polymerase (5’ → 3’) (synthesis of ds-cDNA) ■ Vidarabine: add OH in opposite direction to the normal OH ■ Ara-C: synthetic drug of cytosine arabinoside (cytosine base + arabinose sugar )


Watch the video: 09 Αντιγραφη του DNA Βιολ. Κ., Γ Λυκ. (May 2022).