# Prokaryotic Replication Fork: Mechanism and Key Enzymes

## Introduction to the Prokaryotic Replication Fork

### What is a Replication Fork?

The replication fork is the Y-shaped region of a DNA molecule where active replication is occurring. It forms when the double helix is unwound, exposing the two template strands for new strand synthesis. In prokaryotes, the replication fork is a highly organized molecular machine that moves processively along the chromosome, synthesizing DNA at rates of approximately 500–1000 base pairs per second in *Escherichia coli*.

The fork is not merely a passive opening in the DNA; it is the site of coordinated action by dozens of proteins that unwind, stabilize, prime, synthesize, and proofread DNA. The term "fork" describes the structure: the unreplicated duplex DNA forms the stem, and the two separated single strands form the prongs. Each prong serves as a template for a new complementary strand.

Understanding the replication fork is fundamental to [molecular biology](/blog/careers/molecular-biology) because it is the central structure in genome duplication. Defects in fork components cause replication stress, genome instability, and disease in higher organisms. For prokaryotes, the fork is also a target for antibiotics—ciprofloxacin and other quinolones act by trapping the bacterial enzymes that manage fork progression.

### Origin of Replication and Bidirectional Forks

Prokaryotic replication begins at a single, defined sequence called the origin of replication (*oriC* in *E. coli*). The *oriC* region is approximately 245 base pairs long and contains multiple recognition sites for the initiator protein DnaA. These sites include five DnaA-boxes (9-mer sequences with the consensus TTATCCACA) and an AT-rich region of three 13-mer repeats.

The initiation process follows a defined order:

1. DnaA protein binds to the five DnaA-boxes in an ATP-dependent manner, causing the DNA to wrap around the protein complex.
2. The AT-rich 13-mer repeats are melted (denatured) by the bound DnaA, creating a localized single-stranded bubble.
3. The helicase loader DnaC delivers the DnaB helicase to each of the two single strands in the bubble.
4. DnaB hydrolyzes ATP and begins unwinding the duplex bidirectionally, establishing two replication forks that move in opposite directions around the circular chromosome.

Because the *E. coli* chromosome is circular, the two forks will eventually meet at a region opposite the origin, called the terminus. Bidirectional replication means that the entire chromosome is duplicated in roughly 40 minutes under optimal growth conditions, despite being about 4.6 million base pairs in length. This bidirectionality halves the time required compared to a unidirectional mechanism.

For a visual reference, the [Replication Fork Diagram](/knowledge/molecular-biology/replication-fork-diagram) and [Replication Fork Labeled](/knowledge/molecular-biology/replication-fork-labeled) pages provide annotated illustrations of the structures described throughout this article.

## Key Enzymes and Proteins at the Fork

The replication fork in prokaryotes requires a specific set of proteins, each with a distinct function. The table below summarizes the major components and their roles.

| Protein | Gene | Function | Key Property |
|---------|------|----------|--------------|
| DnaA | *dnaA* | Initiator; melts origin | Binds ATP and origin sequences |
| DnaB | *dnaB* | Helicase; unwinds duplex | 5'→3' translocase; ring-shaped hexamer |
| DnaC | *dnaC* | Helicase loader | Delivers DnaB to origin |
| DnaG | *dnaG* | Primase; synthesizes RNA primers | Requires SSB-coated template |
| SSB | *ssb* | Single-stranded DNA binding protein | Cooperatively coats ssDNA; tetramer |
| DNA Pol III holoenzyme | *dnaE, dnaQ, dnaN, holA-E* | Replicative polymerase | 10 subunits; high processivity |
| DNA Pol I | *polA* | Primer removal; gap filling | 5'→3' polymerase; 5'→3' exonuclease |
| DNA ligase | *ligA* | Seals nicks | Requires NAD+ in *E. coli* |
| Topoisomerase II (gyrase) | *gyrA, gyrB* | Relieves positive supercoils | Introduces negative supercoils |
| Topoisomerase IV | *parC, parE* | Decatenates daughter chromosomes | Separates linked circles |

### Helicase and Unwinding

DnaB is the replicative helicase in *E. coli*. It is a hexameric ring-shaped protein that encircles the lagging strand template and translocates in the 5'→3' direction along that strand. Because the two strands are antiparallel, this movement pushes DnaB toward the fork, where it unwinds the duplex by steric exclusion—the ring physically separates the two strands as it moves.

DnaB requires ATP hydrolysis for translocation. Each subunit of the hexamer binds and hydrolyzes ATP, and the energy from hydrolysis drives conformational changes that move the ring along the DNA. The rate of unwinding by DnaB is approximately 1000 base pairs per second, matching the overall rate of fork movement.

The [Replication Fork Helicase](/knowledge/molecular-biology/replication-fork-helicase) resource provides additional detail on the structure and mechanism of DnaB and related helicases.

### Single-Stranded Binding Proteins

Single-stranded DNA (ssDNA) is thermodynamically unstable and prone to forming secondary structures. It is also vulnerable to nucleases. The single-stranded binding protein (SSB) in *E. coli* is a homotetramer that binds ssDNA cooperatively, coating it in a sequence-independent manner. Each SSB tetramer covers approximately 35 nucleotides.

SSB performs several critical functions at the fork:

- It removes secondary structure from the template strands, keeping them accessible to polymerases.
- It protects ssDNA from degradation by nucleases.
- It stimulates the activity of DnaB helicase and DnaG primase.
- It serves as a protein recruitment platform, interacting with more than a dozen other replication and repair proteins.

The binding of SSB is dynamic; it can slide along the DNA and transfer between binding modes, allowing polymerases to displace it as they synthesize new DNA.

### Primase and RNA Primers

DNA polymerases cannot initiate synthesis de novo; they require a free 3'-OH group to add nucleotides. This limitation is overcome by primase (DnaG), which synthesizes short RNA primers complementary to the template strand.

DnaG is a single polypeptide of approximately 60 kDa. It is recruited to the fork through a direct interaction with DnaB helicase. The primase synthesizes RNA primers of 10–12 nucleotides in length, which are subsequently extended by DNA polymerase III.

Primer synthesis is required on both strands. On the leading strand, a single primer is needed at the origin. On the lagging strand, a new primer is required for each [Okazaki fragment](/knowledge/molecular-biology/okazaki-fragment), meaning DnaG must act repeatedly and processively as the fork advances. The frequency of priming is regulated; primers are synthesized approximately every 1–2 kilobases on the lagging strand.

### DNA Polymerases and Processivity

*E. coli* has five DNA polymerases, but only two are directly involved in replication fork progression: DNA polymerase III (Pol III) and DNA polymerase I (Pol I).

DNA Pol III is the replicative polymerase responsible for bulk DNA synthesis. It is a large, multi-subunit complex (the holoenzyme) with a mass of approximately 900 kDa. The holoenzyme contains:

- Two catalytic cores (αεθ), each containing the polymerase (α, encoded by *dnaE*), the proofreading exonuclease (ε, encoded by *dnaQ*), and a stabilizing subunit (θ, encoded by *holE*).
- Two β-clamps (the processivity factor, encoded by *dnaN*), which tether the polymerase to the DNA.
- A clamp loader complex (γ complex, encoded by *dnaX, holA, holB, holC, holD*), which loads the β-clamps onto DNA.
- The τ (tau) subunits, which dimerize the two catalytic cores and connect them to the helicase.

The β-clamp is a ring-shaped protein that encircles duplex DNA. It slides freely along the DNA but is held at the primer–template junction by the polymerase. This tethering increases the processivity of Pol III from approximately 10 nucleotides (for the core enzyme alone) to more than 50,000 nucleotides. The β-clamp is loaded onto DNA by the clamp loader, which uses ATP hydrolysis to open the ring and place it around the primer–template junction.

DNA Pol I is a monomeric enzyme with three activities: 5'→3' polymerase, 3'→5' exonuclease (proofreading), and 5'→3' exonuclease. Its primary role at the fork is to remove RNA primers and replace them with DNA. The 5'→3' exonuclease activity degrades the RNA primer ahead of the polymerase, while the polymerase fills the resulting gap with DNA. This process is called nick translation.

DNA ligase then seals the remaining nick between the newly synthesized DNA and the adjacent Okazaki fragment. In *E. coli*, DNA ligase uses NAD+ as the energy source for phosphodiester bond formation, a feature unique to bacteria and therefore a target for antibiotics.

## Leading and Lagging Strand Synthesis

The antiparallel nature of DNA creates an inherent asymmetry at the replication fork. DNA polymerases can only synthesize DNA in the 5'→3' direction, meaning they can only add nucleotides to the 3'-OH end of a growing strand. This constraint forces the two template strands to be replicated by different mechanisms.

### Continuous Synthesis on the Leading Strand

The leading strand is synthesized in the same direction as fork movement. Its template strand is oriented 3'→5' relative to the direction of fork progression, allowing the polymerase to synthesize continuously in the 5'→3' direction.

At the fork, the leading strand polymerase is physically tethered to the DnaB helicase through the τ subunit of the Pol III holoenzyme. This tethering ensures that the polymerase moves in lockstep with the helicase, synthesizing DNA at the same rate as the fork advances.

Only one RNA primer is required for leading strand synthesis. This primer is synthesized by DnaG at the origin and is subsequently extended by Pol III. Once initiated, leading strand synthesis continues uninterrupted until the fork reaches the terminus.

### Discontinuous Synthesis and [Okazaki Fragments](/knowledge/molecular-biology/okazaki-fragment)

The lagging strand template is oriented 5'→3' relative to fork movement. Because the polymerase must synthesize in the 5'→3' direction, it must work "backward" relative to the fork, synthesizing short segments in the direction opposite to fork movement.

These segments are called Okazaki fragments, named after their discoverers Reiji and Tsuneko Okazaki. In *E. coli*, Okazaki fragments are typically 1000–2000 nucleotides long.

The synthesis of each Okazaki fragment follows a defined sequence:

1. DnaB helicase unwinds the duplex ahead of the fork, exposing the lagging strand template.
2. SSB coats the exposed single-stranded template.
3. DnaG primase synthesizes a short RNA primer (10–12 nucleotides) at the 3' end of the template region.
4. The clamp loader loads a β-clamp onto the primer–template junction.
5. Pol III extends the primer, synthesizing DNA until it reaches the 5' end of the previous Okazaki fragment.
6. Pol III dissociates, and the cycle repeats for the next fragment.

The lagging strand polymerase must repeatedly dissociate and reassociate as it completes each fragment and moves to a new primer. Despite this discontinuous mechanism, the overall rate of lagging strand synthesis matches that of the leading strand because the polymerase remains physically associated with the replisome.

The [Replication Fork Definition](/knowledge/molecular-biology/replication-fork-definition) page provides a concise summary of these strand-specific mechanisms.

## The Replication Complex and the Replisome

### The Replisome Architecture

The replisome is the complete multiprotein complex that carries out DNA replication at the fork. In *E. coli*, the replisome contains two Pol III holoenzymes (one for each strand), the DnaB helicase, DnaG primase, SSB, and the clamp loader. These components are organized into a single, coordinated machine.

The central organizing element of the replisome is the τ subunit of the Pol III holoenzyme. The τ protein is a product of the *dnaX* gene, which produces both τ and γ subunits through a programmed ribosomal frameshift. The τ subunit performs two critical functions:

- It dimerizes the two catalytic cores of Pol III, holding them together as a single unit.
- It binds directly to DnaB helicase, tethering the polymerases to the unwinding machinery.

The γ subunit is a truncated version of τ that lacks the helicase-binding domain. It is a component of the clamp loader complex, which is responsible for loading β-clamps onto DNA.

The replisome is a highly processive machine. Once assembled at the origin, it can replicate the entire *E. coli* chromosome without dissociating. The processivity of the replisome is remarkable: it synthesizes approximately 4.6 million base pairs in 40 minutes, with an error rate of less than one mistake per billion base pairs.

### Trombone Model and Coordination

The trombone model explains how the replisome coordinates leading and lagging strand synthesis despite the geometric constraints imposed by antiparallel DNA.

In this model, the lagging strand template loops out of the replisome, forming a single-stranded loop that allows the lagging strand polymerase to synthesize DNA in the same physical direction as the leading strand polymerase. The loop grows as the lagging strand polymerase extends an Okazaki fragment, then collapses when the polymerase releases the completed fragment and reinitiates at a new primer.

The name "trombone model" derives from the sliding motion of the loop, which resembles the slide of a trombone. Key features of the model include:

- The two catalytic cores of Pol III are held together by τ, forming a dimeric polymerase.
- The leading strand polymerase is fixed in position, synthesizing continuously.
- The lagging strand polymerase synthesizes DNA on a looping template, repeatedly releasing and rebinding as it completes each Okazaki fragment.
- The loop grows and shrinks in a cyclical manner, coordinating the two polymerases.

The trombone model explains several experimental observations, including the fact that both strands are synthesized at the same rate and that the lagging strand polymerase remains associated with the replisome even when not actively synthesizing DNA.

## Mechanism of Nucleotide Addition and Proofreading

### Nucleotide Incorporation and Base Pairing

DNA polymerase catalyzes the nucleophilic attack of the 3'-OH group of the growing strand on the α-phosphate of an incoming deoxyribonucleoside triphosphate (dNTP). This reaction forms a phosphodiester bond and releases pyrophosphate (PPi). The subsequent hydrolysis of pyrophosphate by inorganic pyrophosphatase makes the reaction effectively irreversible.

The mechanism involves several steps:

1. The polymerase binds a dNTP that is complementary to the template base.
2. The enzyme undergoes a conformational change, closing the active site around the incoming nucleotide.
3. The 3'-OH attacks the α-phosphate, forming the phosphodiester bond.
4. Pyrophosphate is released, and the enzyme returns to the open conformation.
5. The polymerase translocates one base pair along the DNA.

The fidelity of nucleotide selection depends on two factors: the free energy difference between correct and incorrect base pairs, and the ability of the polymerase to discriminate against mismatches. The polymerase active site is shaped to accommodate only Watson–Crick base pairs. Incorrect nucleotides bind with lower affinity and are less likely to induce the conformational change required for catalysis.

The overall error rate of DNA replication in *E. coli* is approximately 10⁻¹⁰ per base pair. This remarkable accuracy is achieved through three layers of proofreading:

- Base selection by the polymerase (error rate ~10⁻⁵)
- Exonucleolytic proofreading (reduces errors to ~10⁻⁷)
- [Mismatch repair](/knowledge/molecular-biology/mismatch-repair) (reduces errors to ~10⁻¹⁰)

### Proofreading by DNA Polymerase III

The ε subunit of Pol III possesses 3'→5' exonuclease activity, which removes misincorporated nucleotides. When the polymerase incorporates an incorrect nucleotide, the resulting mismatch causes the DNA to "fray" at the 3' end. The mismatched base pair is thermodynamically less stable than a correct pair, allowing the DNA to slide back into the exonuclease active site.

The proofreading mechanism operates as follows:

1. The polymerase detects a mismatched base pair.
2. The DNA is transferred from the polymerase active site to the exonuclease active site, a distance of approximately 30 Å.
3. The exonuclease removes the mispaired nucleotide, releasing it as a deoxyribonucleoside monophosphate (dNMP).
4. The DNA slides back to the polymerase active site, and synthesis resumes.

The 3'→5' exonuclease activity is highly selective for mismatched bases. Correctly paired bases are resistant to removal because they remain stably base-paired and do not fray into the exonuclease active site.

The β-clamp plays a critical role in proofreading. It holds the DNA in a position that allows rapid switching between the polymerase and exonuclease active sites. The clamp also prevents the polymerase from dissociating during proofreading, maintaining processivity.

## Termination of Replication and Resolution of Chromosomes

### Termination Sequences and Tus Protein

Replication terminates when the two forks meet at the terminus region, located approximately opposite the origin on the circular chromosome. In *E. coli*, the terminus contains multiple termination sequences called *ter* sites, which are bound by the Tus protein.

The *ter* sites are 23 base pairs in length and are arranged in two clusters with opposite orientations. This arrangement creates a "replication trap" that allows forks to enter the terminus region but prevents them from exiting. The Tus protein binds to *ter* sites and blocks fork progression in a polar manner: a fork approaching from one direction is stopped, while a fork approaching from the other direction is allowed to pass.

The mechanism of fork arrest involves a specific interaction between Tus and the DnaB helicase. When DnaB encounters a Tus–*ter* complex from the blocking direction, it becomes locked onto the DNA. The Tus protein undergoes a conformational change that prevents DnaB from translocating, effectively stalling the fork.

Termination is not perfectly synchronized; one fork typically reaches the terminus before the other. The first fork to arrive stalls at a *ter* site, and the second fork eventually arrives and stalls at the opposite *ter* site. The two forks end up adjacent to each other, leaving a small region of unreplicated DNA between them that is filled in by repair synthesis.

The [Replication Fork Stalling](/knowledge/molecular-biology/replication-fork-stalling) page discusses the consequences of fork arrest and the mechanisms cells use to restart stalled forks.

### Decatenation and Chromosome Segregation

When the two replication forks meet, the daughter chromosomes are topologically linked as catenanes—interlocked circles. This linkage must be resolved before the daughter cells can separate.

Topoisomerase IV, a type II topoisomerase, is responsible for decatenation. Type II topoisomerases cleave both strands of a DNA duplex, pass another duplex through the break, and reseal the cut. Topoisomerase IV specifically recognizes the right-handed crossings between the two daughter chromosomes and removes them, producing two free circular chromosomes.

The decatenation reaction requires ATP hydrolysis and proceeds through a series of steps:

1. Topoisomerase IV binds to a DNA crossing.
2. The enzyme cleaves both strands of one duplex, forming a covalent protein–DNA intermediate.
3. The second duplex is passed through the break.
4. The break is resealed.
5. ATP hydrolysis releases the enzyme for another round of catalysis.

In addition to decatenation, topoisomerase IV also removes precatenanes—intertwines that form behind the fork during replication. These structures arise because the circular chromosome cannot freely rotate to relieve the torsional stress generated by unwinding.

Topoisomerase II (gyrase) also plays a role in replication by removing positive supercoils that accumulate ahead of the fork. Gyrase introduces negative supercoils into DNA, counteracting the positive supercoiling generated by helicase unwinding. Without gyrase, the fork would stall because the DNA ahead of it would become overwound.

## Methods Used to Study the Replication Fork

### In Vitro Replication Assays

The study of the prokaryotic replication fork has been greatly advanced by the development of in vitro replication systems. The most influential of these is the reconstituted *E. coli* replication system, which was developed in the 1970s and 1980s by Arthur Kornberg and colleagues.

The reconstituted system uses purified proteins to replicate plasmids containing the *oriC* origin. The standard reaction mixture includes:

- Template DNA (plasmid containing *oriC*)
- DnaA, DnaB, DnaC (initiation and unwinding)
- DnaG (primase)
- SSB (single-stranded DNA binding)
- Pol III holoenzyme (replicative polymerase)
- Pol I (primer removal)
- DNA ligase (nick sealing)
- Gyrase (topoisomerase)
- dNTPs, ATP, and buffer components

The reaction is typically carried out at 37°C in a buffer containing 20 mM Tris-HCl (pH 7.5), 8 mM magnesium acetate, 50 mM potassium glutamate, and 1 mM DTT. Replication is monitored by measuring the incorporation of radiolabeled dNTPs into acid-precipitable DNA.

In vitro systems allow researchers to:

- Identify the minimal set of proteins required for replication
- Determine the function of individual proteins by omitting them from the reaction
- Measure the kinetics of replication under controlled conditions
- Test the effects of inhibitors and mutations

### Single-Molecule Visualization

Single-molecule techniques have provided unprecedented views of the replication fork in action. These methods allow researchers to observe individual replisomes as they move along DNA, providing information that is obscured in bulk experiments.

One powerful approach is DNA combing, in which DNA molecules are stretched and aligned on a glass surface. Replication origins are labeled with fluorescent nucleotides, and the extent of replication is measured by the length of labeled tracks. This technique is used to measure origin firing and fork rates in populations of cells.

More advanced single-molecule methods include:

- Optical tweezers: A DNA molecule is stretched between two beads, and the replisome is observed as it unwinds the DNA. The force required to stall the fork provides information about the strength of the helicase–polymerase interaction.
- Magnetic tweezers: Similar to optical tweezers but using magnetic beads, allowing measurements of [DNA supercoiling](/knowledge/molecular-biology/dna-supercoiling) during replication.
- Total internal reflection fluorescence (TIRF) microscopy: Fluorescently labeled proteins are observed as they associate with and dissociate from individual DNA molecules. This technique has been used to visualize the dynamics of the β-clamp and clamp loader.

Single-molecule studies have revealed that the replisome is more dynamic than previously thought. The two polymerases do not always move in perfect synchrony; the lagging strand polymerase can pause and restart, and the replisome can transiently dissociate and reassemble without losing its place on the DNA.

## Common Pitfalls and Misconceptions

### Directionality and Antiparallelism

A frequent source of confusion is the relationship between strand directionality and synthesis. Students often struggle with the fact that DNA polymerase synthesizes in the 5'→3' direction, meaning it reads the template in the 3'→5' direction.

A common error is to state that the leading strand is synthesized "in the same direction as the fork" and the lagging strand "in the opposite direction." While this is true, it is more precise to say that the leading strand is synthesized continuously in the direction of fork movement, while the lagging strand is synthesized discontinuously in the opposite direction, producing Okazaki fragments.

Another common misconception is that the leading strand polymerase moves along the template in the 5'→3' direction. This is incorrect. The polymerase moves along the template in the 3'→5' direction (reading the template), but it synthesizes the new strand in the 5'→3' direction. The distinction between template reading and strand synthesis is critical.

### Primer Removal and Gap Filling

Students often confuse the roles of DNA polymerase I and DNA polymerase III. Pol III is the replicative polymerase that synthesizes the bulk of new DNA. Pol I is a repair polymerase that removes RNA primers and fills the resulting gaps.

A common error is to think that Pol I synthesizes Okazaki fragments. It does not. Pol I acts after Pol III has completed an Okazaki fragment, removing the RNA primer and replacing it with DNA. The final nick is sealed by DNA ligase.

Another misconception is that RNA primers are removed by a nuclease and then the gap is filled by a different enzyme. In *E. coli*, Pol I performs both functions through its 5'→3' exonuclease and 5'→3' polymerase activities. The two activities work together in a process called nick translation, in which the enzyme moves along the DNA, removing RNA ahead of it and synthesizing DNA behind it.

The [Replication Fork Short](/knowledge/molecular-biology/replication-fork-short) page provides a concise overview of these concepts for quick review.

## Frequently Asked Questions

### What is the replication fork in prokaryotes?

The replication fork is the Y-shaped structure formed when the double helix is unwound during DNA replication. In prokaryotes, replication begins at a single origin (*oriC* in *E. coli*) and proceeds bidirectionally, creating two forks that move in opposite directions around the circular chromosome. Each fork contains the machinery required for DNA synthesis: helicase, primase, single-stranded binding proteins, and DNA polymerases.

### Which enzymes are involved in the prokaryotic replication fork?

The key enzymes are DnaB helicase (unwinds the duplex), DnaG primase (synthesizes RNA primers), DNA polymerase III holoenzyme (replicative synthesis), DNA polymerase I (primer removal and gap filling), and DNA ligase (nick sealing). Additional proteins include DnaA (initiator), DnaC (helicase loader), SSB (single-stranded DNA binding), and topoisomerases (gyrase and topoisomerase IV) that manage DNA topology.

### Why is the lagging strand synthesized in fragments?

The lagging strand is synthesized in fragments because DNA polymerase can only synthesize in the 5'→3' direction. Since the lagging strand template is oriented 5'→3' relative to fork movement, the polymerase must work backward, synthesizing short segments (Okazaki fragments) that are later joined. Each fragment requires a new RNA primer, and the fragments are sealed by DNA ligase.

### What is the role of RNA primers in DNA replication?

RNA primers provide a free 3'-OH group that DNA polymerase requires to initiate synthesis. DNA polymerase cannot start synthesis de novo; it can only add nucleotides to an existing 3'-OH. Primase (DnaG) synthesizes short RNA primers (10–12 nucleotides) that are subsequently extended by DNA polymerase III. The RNA primers are later removed by DNA polymerase I and replaced with DNA.

### How does DNA polymerase proofread?

DNA polymerase proofreads through its 3'→5' exonuclease activity. When an incorrect nucleotide is incorporated, the mismatch causes the DNA to fray at the 3' end. The DNA is transferred to the exonuclease active site, where the mispaired nucleotide is removed. The DNA then returns to the polymerase active site, and synthesis resumes. This proofreading reduces the error rate from approximately 10⁻⁵ to 10⁻⁷.

### What is the [difference between DNA polymerase I and III](/knowledge/molecular-biology/difference-between-dna-polymerase-i-and-iii)?

DNA polymerase III is the replicative polymerase responsible for bulk DNA synthesis at the fork. It is a large, multi-subunit complex with high processivity, enabled by the β-clamp. DNA polymerase I is a monomeric enzyme that removes RNA primers and fills the resulting gaps. It has 5'→3' polymerase, 3'→5' exonuclease, and 5'→3' exonuclease activities. Pol III synthesizes the majority of new DNA; Pol I acts after Pol III to complete Okazaki fragment processing.

### What is the trombone model?

The trombone model describes how the replisome coordinates leading and lagging strand synthesis. The lagging strand template loops out of the replisome, allowing the lagging strand polymerase to synthesize DNA in the same physical direction as the leading strand polymerase. The loop grows as an Okazaki fragment is extended and collapses when the polymerase releases the completed fragment and reinitiates at a new primer. The two polymerases are held together by the τ subunit of Pol III, ensuring coordinated synthesis.

## Key Takeaways

- The prokaryotic replication fork is a Y-shaped structure formed at the origin of replication (*oriC*), with two forks moving bidirectionally around the circular chromosome.
- The core enzymatic machinery includes DnaB helicase, DnaG primase, SSB, DNA polymerase III holoenzyme, DNA polymerase I, and DNA ligase, each with a specific function.
- The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously as Okazaki fragments, each requiring an RNA primer.
- The replisome coordinates both strands through the τ subunit, which dimerizes the polymerases and tethers them to the helicase, as described by the trombone model.
- DNA polymerase achieves high fidelity through base selection and 3'→5' exonucleolytic proofreading, with the β-clamp maintaining processivity.
- Replication terminates at *ter* sites bound by Tus protein, and daughter chromosomes are resolved by topoisomerase IV decatenation.
- The [Replication Fork Bubble](/knowledge/molecular-biology/replication-fork-bubble) and [Replication Fork Reversal](/knowledge/molecular-biology/replication-fork-reversal) pages provide additional context on fork structure and the cellular responses to replication stress.

## Further Reading

- Mirkin EV, Mirkin SM. *[Replication fork stalling](/knowledge/molecular-biology/replication-fork-stalling) at natural impediments*. Microbiology and [molecular biology](/blog/careers/molecular-biology) reviews : MMBR. 2007. [PubMed 17347517](https://doi.org/10.1128/MMBR.00030-06)
- Lambert S, Carr AM. *Checkpoint responses to replication fork barriers*. Biochimie. 2005. [PubMed 15989976](https://doi.org/10.1016/j.biochi.2004.10.020)
- Bianco PR. *The Biochemical Mechanism of Fork Regression in Prokaryotes and Eukaryotes-A Single Molecule Comparison*. International journal of molecular sciences. 2022. [PubMed 35955746](https://doi.org/10.3390/ijms23158613)
- Thömmes P, Hübscher U. *Eukaryotic DNA replication. Enzymes and proteins acting at the fork*. European journal of biochemistry. 1990. [PubMed 2269294](https://doi.org/10.1111/j.1432-1033.1990.tb19460.x)
- Yeeles JT et al. *Rescuing stalled or damaged replication forks*. Cold Spring Harbor perspectives in biology. 2013. [PubMed 23637285](https://doi.org/10.1101/cshperspect.a012815)
- Labib K, Hodgson B. *Replication fork barriers: pausing for a break or stalling for time?*. EMBO reports. 2007. [PubMed 17401409](https://doi.org/10.1038/sj.embor.7400940)

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* [MAPK Pathway: Mechanism, Function, and Clinical Relevance](/knowledge/molecular-biology/mapk-pathway)
* [Mammalian Cell Culture Bioreactors: A Practical Guide](/knowledge/molecular-biology/mammalian-cell-culture-bioreactor)
* [Nucleotide Formation: Biosynthesis and Assembly of DNA/RNA Building Blocks](/knowledge/molecular-biology/nucleotide-formation)