Semiconservative DNA Replication in Prokaryotes and Eukaryotes: Models, Enzymes, and Proofreading

Table of Contents

• Introduction to Semiconservative DNA Replication
• Enzymes Involved in DNA Replication and Their Functions
• DNA Replication in Prokaryotes
• Proof-reading of replicated DNA
• Evidence for Semi-Conservative Replication of DNA in Prokaryotes
• DNA replication in Eukaryotes
• Proofreading
• Evidence for Semi-Conservative Replication of DNA in Eukaryotes
• Difference between Prokaryotic and Eukaryotic Replication
• References

Introduction to Semiconservative DNA Replication

• In the semiconservative model of DNA replication, two identical copies of the original DNA molecule are produced, each containing one original (parental) strand and one newly synthesized strand.
• Watson and Crick’s double helix model of DNA includes an integrated template system that enables self-replication or autocatalysis.
• Due to specific base pairing—Adenine with Thymine, and Guanine with Cytosine—the sequence of bases on one strand determines the sequence on the complementary strand.
• Each strand of the DNA double helix serves as a template for synthesizing its complementary strand.
• Watson and Crick proposed that replication begins with the disruption of hydrogen bonds between base pairs, followed by the rotation and separation of the two polynucleotide strands.
• Each purine and pyrimidine base on the parent strand attracts its complementary free nucleotide in the cell, holding it in place through specific hydrogen bonding.
• Once held in place, the free nucleotides are joined together by phosphodiester bonds that link adjacent deoxyribose residues, creating a new polynucleotide strand with a specific base sequence.
• The outcome of replication is two double-stranded DNA molecules with base sequences identical to the original DNA.
• One daughter DNA molecule includes the original left strand, while the other includes the original right strand.
• Because each daughter molecule retains half of the original DNA, this pattern of replication is termed semiconservative.
• DNA replication occurs in both prokaryotes and eukaryotes, and while the fundamental mechanism is similar, the process is more complex in eukaryotes.
• Besides the semiconservative model, two other possible models of DNA replication have been proposed:
• In the conservative model, both newly synthesized strands form the new DNA molecule, while the two original strands remain together, fully conserved.
• In the dispersive model, the parent DNA double helix is broken into fragments during replication, and the new DNA molecules consist of interspersed segments of parental and newly synthesized DNA.

Enzymes Involved in DNA Replication and Their Functions

• DNA Helicase: Unwinds the DNA helix by breaking hydrogen bonds between bases.
• Topoisomerase: Alleviates tension in the DNA strand by cutting and resealing it during unwinding.
• Single-strand binding (SSB) proteins: Prevent the single-stranded DNA from reannealing after being separated.
• Primase: Synthesizes short RNA primers to initiate DNA synthesis.
• DNA Polymerase I: Removes RNA primers and fills in the gaps with DNA.
• DNA Polymerase II: Mainly functions in DNA repair mechanisms.
• DNA Polymerase III (α, δ, ε): Primary enzyme for adding nucleotides during replication.
• Sliding Clamp: Stabilizes DNA polymerase on the DNA strand for efficient replication.
• DNA Ligase: Joins Okazaki fragments on the lagging strand to form a complete DNA molecule.

DNA Replication in Prokaryotes

DNA replication in prokaryotes occurs in three main stages:

1. Initiation
2. Elongation
3. Termination

Initiation of DNA Replication in Prokaryotes

• Replication starts at a specific location on the chromosome known as the origin of replication. In Escherichia coli (and most other prokaryotes), this origin is referred to as OriC.
• OriC is approximately 245 base pairs long and contains multiple AT-rich sequences, which are easier to unwind due to having only two hydrogen bonds.
• This region includes repeated sequences—specifically, 13 bp repeats and 9 bp repeats.
• Initially, around 30 DnaA proteins bind to the 9 bp repeats, causing the DNA to bend and initiate the unwinding of the helix at the adjacent 13 bp repeats.
• DnaC proteins then help load DNA helicase (DnaB) onto the origin.
• DNA helicase unwinds the DNA by breaking the hydrogen bonds between base pairs, a process that requires ATP hydrolysis.
• As the DNA unwinds, replication forks—Y-shaped structures—are formed. Two replication forks are established and proceed bidirectionally.
• The activity of DNA helicase generates topological stress, causing the formation of supercoiled DNA.
• Topoisomerase resolves this stress by temporarily cutting the DNA helix and resealing it, preventing overwinding ahead of the replication fork.
• Single-strand binding proteins (SSBs) bind to the separated strands to stabilize them and prevent re-annealing.
• DNA polymerase cannot initiate synthesis independently—it can only extend a chain in the 5′ to 3′ direction and needs a free 3′-OH group for nucleotide addition.
• The enzyme RNA primase synthesizes a short RNA primer (5–10 nucleotides) complementary to the DNA strand to provide the required 3′-OH group.

Elongation of DNA Replication in Prokaryotes

• DNA polymerase III is loaded onto the DNA, initiating synthesis.
• The two DNA strands have opposite orientations: one runs 5′ to 3′, and the other 3′ to 5′.
• The strand complementary to the 3′ to 5′ parental strand is synthesized continuously toward the replication fork and is called the leading strand; it requires only one primer.
• The other strand, complementary to the 5′ to 3′ template, is synthesized in short stretches known as Okazaki fragments and is referred to as the lagging strand. This strand requires multiple primers.
• A sliding clamp protein ensures that DNA polymerase remains attached to the DNA during synthesis.
• DNA polymerase I removes RNA primers using its exonuclease activity and replaces them with DNA nucleotides.
• DNA ligase then seals the remaining gaps between fragments by forming phosphodiester bonds.
• The two replication forks continue to move in opposite directions around the circular chromosome.

Termination of DNA Replication in Prokaryotes

• Termination occurs when the two replication forks meet, resulting in two complete, double-stranded DNA molecules.
• A termination protein (Tus) binds to specific ter sequences to block one of the replication forks and halt further progression of DNA helicase.
• The second fork stops when it encounters the first.
• During replication, the circular bacterial chromosome can become entangled or linked due to crossover events.
• Topoisomerase IV separates these interlinked DNA circles (a process called decatenation).
• Finally, the two DNA copies are distributed into two daughter cells during cell division.

Proof-reading of replicated DNA

• Proofreading of replicated DNA involves scanning the ends (termini) of newly synthesized DNA chains to detect and correct errors during the replication process.
• This proofreading mechanism is carried out by the 3′ to 5′ exonuclease activity of DNA polymerase.
• It functions by removing incorrectly paired nucleotides at the 3′ end of the growing DNA strand, allowing the 5′ to 3′ polymerase activity to then add the correct nucleotide.
• In bacteria, all three DNA polymerases—Pol I, Pol II, and Pol III—are capable of proofreading through their 3′ to 5′ exonuclease activity.
• When DNA polymerase detects a mismatched base pair, it reverses direction by one base pair, excises the incorrect nucleotide, and then resumes forward synthesis after inserting the correct base.
• The efficiency of proofreading directly influences the mutation rate during DNA replication.

Evidence for Semi-Conservative Replication of DNA in Prokaryotes

• The semiconservative nature of DNA replication in prokaryotes was experimentally confirmed in 1958 by Matthew Meselson and Franklin W. Stahl.
• They used isotopically labeled DNA and an isopycnic density gradient centrifugation technique to demonstrate this.
• Escherichia coli was grown for several generations in a medium containing ammonium chloride (NH₄Cl) with ¹⁵N (a heavy isotope of nitrogen).
• When DNA was isolated from these cells and centrifuged in a cesium chloride (CsCl) salt density gradient, it separated at the point where its density equaled that of the solution.
• The DNA from ¹⁵N-grown cells was denser than DNA from cells grown in normal ¹⁴N medium.
• The ¹⁵N-labeled E. coli cells were then transferred to a ¹⁴N medium and allowed to divide.
• Cell division was tracked using microscopic counts and colony assays.
• DNA was extracted at various time intervals and compared to pure ¹⁴N DNA and pure ¹⁵N DNA.
• After one round of replication, the extracted DNA showed an intermediate density.
• This intermediate result ruled out conservative replication, which would have resulted in two distinct bands: one for heavy DNA (¹⁵N) and one for light DNA (¹⁴N), with no intermediate band.
• However, both semiconservative and dispersive replication could account for the intermediate density at this stage.
• In semiconservative replication, each DNA molecule would consist of one ¹⁵N strand and one ¹⁴N strand, producing intermediate density.
• In dispersive replication, each DNA strand would contain segments of both ¹⁵N and ¹⁴N, also resulting in intermediate density.
• After two rounds of replication, the DNA separated into two distinct bands: one at intermediate density and one at the lighter ¹⁴N density.
• This observation did not support dispersive replication, which would have resulted in a single, slightly lighter band due to uniform dilution of ¹⁵N in all DNA strands.
• The presence of two bands—one intermediate and one light—confirmed semiconservative replication, where half the DNA molecules had one old (¹⁵N) and one new (¹⁴N) strand, and the other half had two new (¹⁴N) strands.

DNA replication in Eukaryotes

DNA replication in eukaryotes takes place in three main stages:

1. Initiation
2. Elongation
3. Termination

Initiation of DNA replication in Eukaryotes

• Eukaryotic chromosomes have multiple origins of replication; humans may have up to 100,000 origins.
• At each origin, a pre-replication complex (pre-RC) is formed during the G1 phase of the cell cycle.
• The pre-RC assembly includes:
• Six Origin Recognition Complex (ORC) proteins (ORC1–6),
• Cdc6 (Cell division control protein),
• Cdt1 (Chromatin Licensing and DNA Replication Factor),
• Six Mini Chromosome Maintenance (MCM2–7) proteins.
• Once the pre-RC is in place, two kinases activate it:
• Cyclin-dependent kinase 2 (CdK),
• Dbf4-dependent kinase (DdK).
• These kinases recruit Cdc45, which in turn brings in other DNA replication proteins.
• Cdc45, MCM2–7, and the GINS complex (Go-Ichi-Ni-San) combine to form the CMG helicase, which becomes active in the S phase.
• CMG helicase unwinds the origin of replication, forming replication forks.
• Each origin of replication forms two replication forks, which progress bidirectionally.
• The action of helicase creates supercoiled DNA, inducing topological stress.
• Topoisomerase and Replication Factor A (RF-A) relieve this stress by altering DNA topology.
• Single-strand binding proteins stabilize unwound DNA and prevent it from re-annealing.
• Primase, tightly associated with DNA polymerase α, synthesizes short RNA primers to begin DNA synthesis.

Elongation of DNA replication in Eukaryotes

• Upon entering the S phase, the replication complex transitions into the replisome, coordinating DNA replication.
• DNA polymerase ε synthesizes the leading strand continuously, in the same direction as the unwinding.
• DNA polymerase α initiates Okazaki fragments on the lagging strand by synthesizing 20–30 nucleotides.
• These fragments are further extended by DNA polymerase δ, synthesizing DNA in a discontinuous manner.
• PCNA (Proliferating Cell Nuclear Antigen) acts as a sliding clamp, holding the DNA polymerases in place during synthesis.
• RNase H removes most of the RNA primer, but one ribonucleotide remains attached at the 3′ end.
• Flap endonuclease 1 (FEN 1) removes this remaining ribonucleotide.
• DNA polymerase δ fills in the gaps between Okazaki fragments after primer removal.
• DNA ligase seals the nicks between fragments, creating a continuous lagging strand.

Termination of DNA replication in Eukaryotes

• Termination of replication occurs when two replication forks converge.
• The exact location of fork convergence is not predetermined, as it depends on the timing and speed of fork progression.
• When forks meet, torsional strain builds up behind them and is resolved by Topoisomerase II.
• The CMG helicase is thought to slide onto the last Okazaki fragment’s double-stranded DNA during termination.
• A key step in termination is the removal of CMG helicase from chromatin.
• This is achieved through ubiquitination of the MCM7 subunit, followed by disassembly of the replisome by Cdc48.
• Because eukaryotic chromosomes are linear, replication cannot reach the very ends of chromosomes.
• This is due to the need for an RNA primer on the lagging strand, which causes the loss of a DNA segment with every cycle.
• To prevent gene loss, telomeres—repetitive, non-coding sequences—are located at the chromosome ends.
• In humans, this telomeric sequence is TTAGGG, repeated 100–1000 times.
• Telomeres protect coding DNA, and only these non-essential sequences are lost during replication.
• Eventually, telomere shortening limits the number of times a cell can divide, a threshold known as the Hayflick limit.
• In germ cells, certain stem cells, and some white blood cells, telomerase enzyme extends the telomeres to prevent DNA degradation.
• Somatic cells, however, do not express telomerase, so their telomeres shorten with each replication cycle.
• Uncontrolled activation of telomerase in somatic cells can lead to cancer, allowing these cells to divide indefinitely.

Proofreading

• Three replicative DNA polymerases—Pol α, Pol δ, and Pol ɛ—are responsible for replicating the eukaryotic genome.
• Pol α lacks internal proofreading activity, resulting in low fidelity during DNA synthesis.
• With an estimated base substitution error rate of 10⁻⁴, Pol α contributes to the replication of approximately 1.5% of the eukaryotic genome, introducing hundreds of mismatches in each replication cycle.
• Pol δ has exonucleolytic proofreading ability, which allows it to correct errors made by Pol α.
• Errors introduced by Pol δ and Pol α cannot be corrected by Pol ɛ.
• However, Pol δ can also proofread and correct errors made by Pol ɛ on the leading strand during replication.

Evidence for Semi-Conservative Replication of DNA in Eukaryotes

• In 1957, J.H. Taylor and P. Woods provided evidence for the semi-conservative replication of DNA in eukaryotes.
• They conducted their experiment using root tip cells of the broad bean plant (Vicia faba).
• The technique involved splitting root tip cells and using autoradiography along with light microscopy.
• To label the DNA, the cells were treated with radioactive thymidine (³H-thymidine), which incorporated into newly synthesized DNA strands.
• After labeling, the cells were transferred to a normal (unlabeled) growth medium containing colchicine, which inhibits sister chromatid separation during anaphase.
• The use of colchicine ensured that both sister chromatids remained together, allowing for easier observation of DNA distribution.
• In the first generation, both DNA strands showed even distribution of radioactivity, with one radioactively labeled strand and one unlabeled strand per DNA molecule.
• In the second generation, only one chromatid of each chromosome displayed radioactivity.
• This pattern confirmed that only one strand of the DNA in each new molecule came from the original labeled DNA, while the other was newly synthesized.
• These results supported the semi-conservative model of DNA replication, showing that each daughter DNA molecule inherits one old (parental) strand and one newly synthesized strand.

Difference between Prokaryotic and Eukaryotic Replication

Origin of replication

• Prokaryotes: Single origin per DNA molecule
• Eukaryotes: Multiple origins per chromosome

Location

• Prokaryotes: Replication occurs in the cytoplasm
• Eukaryotes: Replication occurs in the nucleus

Rate of replication

• Prokaryotes: Faster (≈1000 nucleotides per second)
• Eukaryotes: Slower (≈50–100 nucleotides per second)

DNA polymerase types

• Prokaryotes: 5 types
• Eukaryotes: 14 types

Telomerase

• Prokaryotes: Not present
• Eukaryotes: Present to maintain telomere length

RNA primer removal

• Prokaryotes: Done by DNA polymerase I
• Eukaryotes: Done by RNase H

Strand elongation

• Prokaryotes: By DNA polymerase III
• Eukaryotes: By Pol α, Pol δ, and Pol ε

Sliding clamp

• Prokaryotes: Sliding clamp
• Eukaryotes: PCNA (Proliferating Cell Nuclear Antigen)

References

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• Taylor, J. H., Woods, P. S., & Hughes, W. L. (1957). The organization and duplication of chromosomes as revealed by autoradiographic studies using tritium-labeled thymidine. Proceedings of the National Academy of Sciences of the United States of America, 43(1), 122–128. https://doi.org/10.1073/pnas.43.1.122
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