# Replication Forks and Lagging Strand Synthesis Explained

## Introduction to Replication Forks and the Lagging Strand

DNA replication is the process by which a cell duplicates its entire genome before division. This process is carried out at specialized structures called **replication forks**, which are Y-shaped regions where the double helix is actively unwound and new DNA is synthesized. The term [Replication Fork Definition](/knowledge/molecular-biology/replication-fork-definition) refers to the dynamic junction between the unreplicated parental duplex and the two daughter strands being produced. At each fork, the parental DNA serves as a template for the synthesis of two new strands, following the rules of Watson–Crick base pairing.

The fundamental challenge of DNA replication arises from the antiparallel nature of the DNA double helix. The two parental strands run in opposite directions: one in the 5′ to 3′ direction and the other in the 3′ to 5′ direction. All DNA polymerases synthesize new DNA exclusively in the 5′ to 3′ direction, adding nucleotides to the 3′-hydroxyl (OH) group of the growing strand. This creates an intrinsic asymmetry at the replication fork that cells must resolve through a sophisticated division of labor between the leading and lagging strands.

### The Antiparallel Challenge

Consider a replication fork moving in a given direction along the parental DNA. One template strand is oriented such that its 3′ end is nearest the fork. DNA polymerase can read this template in the 3′ to 5′ direction and synthesize a complementary strand continuously in the 5′ to 3′ direction, moving in the same direction as the fork. This is the **leading strand**.

The other template strand is oriented in the opposite direction, with its 5′ end nearest the fork. To synthesize a complementary strand in the 5′ to 3′ direction, DNA polymerase would have to move in the direction opposite to fork progression. This is physically impossible because the polymerase would be moving away from the unwound region. The solution is that this strand, called the **lagging strand**, is synthesized discontinuously in short segments. Each segment is synthesized in the 5′ to 3′ direction, but the polymerase repeatedly dissociates and reassociates as the fork advances, creating a series of fragments that are later joined together.

### Leading vs. Lagging Strand

The distinction between leading and lagging strand synthesis is not based on any difference in the polymerase enzymes themselves—both strands are synthesized by the same DNA polymerase III holoenzyme in bacteria. Rather, the difference lies in the **continuity** of synthesis. The leading strand is synthesized as one continuous molecule from a single RNA primer. The lagging strand is synthesized as a series of short DNA fragments, each initiated by its own RNA primer. These fragments are called **[Okazaki fragments](/knowledge/molecular-biology/okazaki-fragment)**, named after Reiji and Tsuneko Okazaki, who discovered them in the 1960s.

In bacteria, Okazaki fragments are typically 1,000 to 2,000 nucleotides long. In eukaryotes, they are much shorter, typically 100 to 200 nucleotides. The existence of these fragments is direct evidence of the discontinuous nature of lagging strand synthesis. Understanding the machinery that creates and joins these fragments is central to understanding DNA replication as a whole.

## The Replication Fork Machinery

The replication fork is not simply a bare DNA template; it is a highly organized protein complex called the **replisome**. This multi-protein machine coordinates the unwinding of the parental duplex with the synthesis of both daughter strands. The replisome includes helicases, primases, polymerases, sliding clamps, and clamp loaders, each performing a specific function.

### Helicase and Unwinding

The first step at any replication fork is the separation of the two parental strands. This is accomplished by a **DNA helicase**, a motor protein that uses the energy of ATP hydrolysis to break the hydrogen bonds between base pairs and processively unwind the duplex. In *E. coli*, the replicative helicase is **DnaB**, a hexameric ring-shaped protein that translocates along single-stranded DNA in the 5′ to 3′ direction. DnaB encircles the lagging strand template and pushes through the duplex, unwinding it ahead of the polymerases.

Helicase action creates two types of single-stranded DNA (ssDNA) regions: the leading strand template, which is exposed in the 3′ to 5′ direction, and the lagging strand template, which is exposed in the 5′ to 3′ direction. These single-stranded regions are immediately coated by **single-stranded DNA binding proteins (SSBs)** in bacteria (called RPA, replication protein A, in eukaryotes). SSBs protect the exposed template from nucleases and prevent the formation of secondary structures that would impede polymerase progression.

The unwinding of the double helix generates torsional stress ahead of the fork. As the helicase unwinds DNA, it introduces positive supercoils ahead of the replication fork. These are relieved by **topoisomerases**, enzymes that transiently break and reseal the DNA backbone. In bacteria, DNA gyrase (a type II topoisomerase) removes positive supercoils ahead of the fork. Without topoisomerase activity, the accumulating supercoils would eventually stall the helicase and block replication entirely.

### Primase and RNA Primers

DNA polymerases cannot initiate synthesis *de novo*; they require a free 3′-OH group to which they can add nucleotides. This requirement is met by **primase**, an RNA polymerase that synthesizes short RNA primers complementary to the DNA template. Primase is a specialized enzyme that can initiate synthesis without a primer, using ribonucleotide triphosphates (NTPs) as substrates.

In *E. coli*, the primase is the **DnaG** protein. DnaG interacts directly with the DnaB helicase at the fork and synthesizes RNA primers of approximately 10 to 12 nucleotides in length. The leading strand requires only a single RNA primer at the origin of replication. The lagging strand, however, requires a new RNA primer for every Okazaki fragment. This means primase must act repeatedly at the fork, synthesizing a new primer approximately every 1 to 2 seconds in bacteria.

The requirement for RNA primers is a universal feature of DNA replication. The [Primase Work on the Lagging Strand](/knowledge/molecular-biology/primase-work-on-the-lagging-strand) is particularly demanding because it must be coordinated with the periodic release and reassociation of the lagging strand polymerase. The RNA primers are later removed and replaced with DNA, as described in the section on Okazaki fragment maturation.

### DNA Polymerase III and the Sliding Clamp

The main replicative polymerase in *E. coli* is **DNA polymerase III holoenzyme**. This large complex (approximately 900 kDa) contains two catalytic cores, one for the leading strand and one for the lagging strand, held together by a dimeric clamp loader complex. Each catalytic core contains the α subunit (polymerase activity), the ε subunit (3′ to 5′ exonuclease proofreading activity), and the θ subunit (stimulates ε).

DNA polymerase III has two critical properties that make it suitable for replication. First, it is highly **processive**, meaning it can add thousands of nucleotides without dissociating from the template. This processivity is conferred by the **β sliding clamp**, a ring-shaped protein that encircles the DNA template and tethers the polymerase to it. The β clamp is loaded onto the DNA by the γ complex (the clamp loader), which uses ATP hydrolysis to open the ring and place it around the primer-template junction.

Second, DNA polymerase III has **3′ to 5′ exonuclease activity**, which allows it to proofread. When the polymerase misincorporates a nucleotide, the mismatched base pair causes the polymerase to pause and the nascent 3′ end to fray into the exonuclease active site. The incorrect nucleotide is removed, and synthesis resumes. This proofreading activity reduces the error rate from approximately 10⁻⁴ (without proofreading) to approximately 10⁻⁶ to 10⁻⁷ (with proofreading).

The sliding clamp is not unique to bacteria. Eukaryotes use **PCNA** (proliferating cell nuclear antigen) as their sliding clamp, and the clamp loader is **RFC** (replication factor C). The fundamental mechanism is conserved: a ring-shaped protein encircles the DNA, and a loader places it at the primer-template junction.

## Mechanism of Lagging Strand Synthesis

The synthesis of the lagging strand is a cyclical process that involves repeated primer synthesis, fragment elongation, primer removal, gap filling, and ligation. Each cycle produces one Okazaki fragment. Understanding this cycle requires following the events in a precise order.

### Okazaki Fragment Formation

The formation of an Okazaki fragment begins when the DnaB helicase unwinds a new stretch of the lagging strand template, exposing a single-stranded region. The DnaG primase, which is associated with DnaB, synthesizes a short RNA primer complementary to the template. This primer provides the free 3′-OH group required by DNA polymerase III.

Once the primer is laid down, the clamp loader (γ complex) loads a β clamp onto the primer-template junction. DNA polymerase III then binds to the clamp and begins adding deoxyribonucleotides to the 3′ end of the primer, extending it in the 5′ to 3′ direction. The polymerase continues synthesis until it reaches the 5′ end of the previous Okazaki fragment, at which point it dissociates.

The length of an Okazaki fragment is determined by the frequency of primer synthesis, which in turn is determined by the rate of helicase unwinding and the processivity of the polymerase. In *E. coli*, the fork moves at approximately 1,000 nucleotides per second, and Okazaki fragments are 1,000 to 2,000 nucleotides long, meaning a new primer is synthesized every 1 to 2 seconds.

The key point is that each Okazaki fragment begins with an RNA primer at its 5′ end. This RNA must be removed and replaced with DNA before the fragments can be joined. The steps of this maturation process are described below.

### Primer Removal and Gap Filling

The RNA primers at the 5′ ends of Okazaki fragments must be removed and replaced with DNA. In *E. coli*, this is accomplished by **DNA polymerase I**, a different enzyme from the replicative polymerase III. DNA polymerase I has three activities: 5′ to 3′ polymerase, 3′ to 5′ exonuclease (proofreading), and 5′ to 3′ exonuclease. The 5′ to 3′ exonuclease activity is unique to polymerase I and is used to remove RNA primers.

The process works as follows. When DNA polymerase III reaches the 5′ end of the previous Okazaki fragment, it stalls and dissociates. DNA polymerase I then binds at the nick—the junction between the RNA primer of the downstream fragment and the DNA of the upstream fragment. The 5′ to 3′ exonuclease activity of polymerase I removes the RNA nucleotides one at a time, while the polymerase activity simultaneously adds DNA nucleotides to the 3′ end of the upstream fragment, filling the gap. This coupled removal and synthesis is called **nick translation**.

The result is a continuous DNA strand with a single nick—a missing phosphodiester bond between the 3′-OH of the newly added DNA and the 5′-phosphate of the upstream fragment. This nick is sealed by DNA ligase.

### Ligation by DNA Ligase

**DNA ligase** catalyzes the formation of a phosphodiester bond between the 3′-OH of one nucleotide and the 5′-phosphate of the adjacent nucleotide. In *E. coli*, DNA ligase uses NAD⁺ as an energy source, while eukaryotic ligases use ATP. The enzyme first forms a covalent ligase-AMP intermediate, then transfers the AMP to the 5′-phosphate at the nick, activating it. The 3′-OH of the adjacent nucleotide then attacks the activated phosphate, forming the phosphodiester bond and releasing AMP.

The ligation step completes the Okazaki fragment maturation process. After ligation, the lagging strand is a continuous DNA molecule, indistinguishable from the leading strand. Defects in DNA ligase are lethal because unrepaired nicks accumulate and the lagging strand remains fragmented.

The complete cycle of Okazaki fragment synthesis and maturation can be summarized as follows:

1. DnaB helicase unwinds the parental duplex, exposing the lagging strand template.
2. DnaG primase synthesizes a short RNA primer (10–12 nucleotides) at the 5′ end of the new fragment.
3. The γ complex loads a β clamp onto the primer-template junction.
4. DNA polymerase III binds the clamp and extends the primer in the 5′ to 3′ direction.
5. Synthesis continues until the polymerase reaches the 5′ end of the previous Okazaki fragment.
6. DNA polymerase III dissociates, and DNA polymerase I removes the RNA primer and fills the gap with DNA.
7. DNA ligase seals the nick between adjacent fragments.

## Coordination of Leading and Lagging Strand Synthesis

The leading and lagging strand polymerases must work at the same rate to avoid one strand getting ahead of the other. This coordination is achieved through a remarkable structural arrangement known as the **trombone model**, first proposed by Bruce Alberts in the 1980s.

### The Trombone Model

In the trombone model, the two catalytic cores of DNA polymerase III holoenzyme are physically linked together by the τ (tau) subunits of the clamp loader. Both polymerases are thus part of a single complex that moves as a unit along the DNA. The leading strand polymerase tracks along the leading strand template, synthesizing DNA continuously in the direction of fork movement.

The lagging strand polymerase, however, is oriented in the opposite direction. It synthesizes DNA away from the fork, on the looped-out lagging strand template. As the helicase unwinds more DNA, the lagging strand template is extruded as a single-stranded loop. The lagging strand polymerase synthesizes an Okazaki fragment while moving along this loop, and when it reaches the previous fragment, it releases the template and the loop collapses.

The loop grows and shrinks like the slide of a trombone, giving the model its name. This arrangement allows both polymerases to move in the same physical direction (with the fork) while synthesizing DNA in opposite chemical directions (5′ to 3′ for both, but on opposite template strands).

### Looping of the Lagging Strand

The looping of the lagging strand template is a direct consequence of the trombone model. As the DnaB helicase unwinds the duplex, the lagging strand template is extruded in the 5′ to 3′ direction. Because the lagging strand polymerase is moving away from the fork, the template must loop back on itself to reach the polymerase active site.

The size of the loop varies throughout the Okazaki fragment synthesis cycle. At the start of a new fragment, the loop is small. As the polymerase extends the fragment, the loop grows. When the polymerase reaches the previous fragment and dissociates, the loop collapses, and a new loop begins to form for the next fragment.

This looping mechanism has several important consequences. First, it ensures that both polymerases are physically coupled, so they advance at the same rate. Second, it allows the lagging strand polymerase to remain associated with the replisome even though it repeatedly dissociates from the DNA template. Third, it explains why Okazaki fragments are synthesized in a specific size range—the size is determined by the length of the loop that can be accommodated before the polymerase must release.

The trombone model has been supported by direct observation. Single-molecule fluorescence studies have visualized the looping of the lagging strand template in real time, confirming that the loop grows and collapses in synchrony with Okazaki fragment synthesis.

## Experimental Evidence for Discontinuous Synthesis

The discovery that DNA replication is discontinuous on one strand was one of the most important findings in molecular biology. The evidence came from a series of elegant experiments performed in the 1960s.

### Okazaki's Pulse-Chase Experiments

Reiji Okazaki and his colleagues performed the key experiments demonstrating discontinuous synthesis. They grew *E. coli* cells and added radioactive thymidine for very short periods (a "pulse" of a few seconds). They then immediately extracted the DNA and analyzed it by sedimentation through alkaline sucrose gradients.

The results were striking. After a short pulse, most of the radioactivity was found in short DNA fragments, approximately 1,000 to 2,000 nucleotides in length. These fragments were named **Okazaki fragments**. When the pulse was followed by a "chase" with unlabeled thymidine for a longer period, the radioactivity was progressively found in larger DNA molecules, indicating that the short fragments were being joined together.

These experiments provided direct evidence that at least one strand of DNA is synthesized discontinuously. The short fragments observed after a brief pulse represent the Okazaki fragments that have not yet been joined. The chase experiments showed that these fragments are precursors to full-length DNA.

### Electron Microscopy of Replication Forks

Additional evidence came from electron microscopy studies of replicating DNA. When replication forks were visualized under the electron microscope, researchers observed single-stranded regions on one side of the fork—the lagging strand template. These single-stranded regions were consistent with the idea that the lagging strand is synthesized discontinuously, leaving transient gaps between Okazaki fragments.

Electron microscopy also revealed the presence of replication "bubbles" and "eyes" in replicating DNA, consistent with bidirectional replication from a single origin. These observations, combined with the biochemical evidence from Okazaki's experiments, firmly established the discontinuous model of lagging strand synthesis.

The [Semiconservative Replication](/knowledge/molecular-biology/semiconservative-replication) model, established by Meselson and Stahl in 1958, provided the framework for understanding how the two strands are distributed to daughter molecules. The Okazaki experiments added the crucial detail that one of these strands is synthesized in pieces.

## Methods to Study Replication Forks and Lagging Strand

Modern molecular biology has developed powerful techniques to study replication forks and lagging strand synthesis in detail. These methods allow researchers to visualize replication dynamics at the single-molecule level and to map replication events across entire genomes.

### DNA Fiber Analysis

**DNA fiber analysis** (also called DNA combing) is a technique for visualizing replication forks on individual DNA molecules. Cells are labeled with two different thymidine analogs, such as iododeoxyuridine (IdU) followed by chlorodeoxyuridine (CldU). The labeled DNA is then stretched on a glass slide and detected with fluorescent antibodies specific for each analog.

The resulting "fiber" shows a series of fluorescent tracks. The length of each track corresponds to the amount of DNA synthesized during the labeling period, and the pattern of tracks reveals the direction and speed of fork movement. This technique can be used to measure fork speed, determine the spacing between origins, and detect [replication fork stalling](/knowledge/molecular-biology/replication-fork-stalling) or collapse.

### Single-Molecule Fluorescence

Single-molecule [fluorescence microscopy](/knowledge/diagnostics/imaging/fluorescence-microscopy-principles-applications-and-image-acquisition) has revolutionized the study of replication forks. By fluorescently labeling individual replisome components, researchers can observe the dynamics of the replication machinery in real time. For example, studies using total internal reflection fluorescence (TIRF) microscopy have directly visualized the trombone model loop, showing the growth and collapse of the lagging strand loop during Okazaki fragment synthesis.

These studies have revealed that the replisome is remarkably dynamic. Individual polymerases can exchange with free polymerase in solution, and the clamp loader can load new clamps onto the DNA while the replisome is actively synthesizing. Single-molecule studies have also shown that replication forks can pause, restart, and even reverse under certain conditions.

### Genomic Approaches

Next-generation sequencing has enabled genome-wide analysis of replication. **Replication timing analysis** uses sequencing to determine when each region of the genome is replicated during S phase. **Okazaki fragment mapping** uses deep sequencing of the short fragments to identify the positions of RNA primers and the sites of Okazaki fragment junctions.

These genomic approaches have revealed that Okazaki fragment junctions are not randomly distributed. In eukaryotes, they are enriched at specific sequence motifs and are influenced by [chromatin structure](/knowledge/molecular-biology/chromatin-structure). The analysis of Okazaki fragments has also provided insights into the mechanisms of primer removal and the coordination of leading and lagging strand synthesis.

## Common Pitfalls and Misconceptions

Students frequently encounter several conceptual difficulties when learning about replication forks and lagging strand synthesis. Understanding these common errors can help clarify the underlying biology.

### Directionality Errors

The most common error is confusing the direction of DNA synthesis with the direction of fork movement. DNA polymerase always synthesizes in the 5′ to 3′ direction. The leading strand is synthesized in the same direction as fork movement, while the lagging strand is synthesized in the opposite direction. However, both strands are synthesized by polymerases moving in the 5′ to 3′ direction along their respective templates.

A related error is thinking that the lagging strand polymerase moves "backward" along the DNA. In the trombone model, the lagging strand polymerase is physically moving forward with the replisome, but it is synthesizing DNA on a looped template. The polymerase itself does not move backward; the template loops out to accommodate the opposite direction of synthesis.

### Confusing Primase with Polymerase

Another common error is confusing primase with DNA polymerase. Primase is an RNA polymerase, not a DNA polymerase. It synthesizes short RNA primers using ribonucleotide triphosphates (NTPs), not deoxyribonucleotide triphosphates (dNTPs). The RNA primers are later removed and replaced with DNA by DNA polymerase I.

Students also sometimes think that the leading strand does not require a primer. This is incorrect. Both strands require RNA primers. The leading strand requires only one primer at the origin, while the lagging strand requires a new primer for each Okazaki fragment.

### Supercoiling and Topoisomerase

A third common misconception involves the role of topoisomerase. Some students think that topoisomerase is required to unwind the DNA at the fork. This is incorrect. The helicase unwinds the DNA. Topoisomerase relieves the torsional stress (supercoiling) that accumulates ahead of the fork as a result of unwinding.

Without topoisomerase, the positive supercoils ahead of the fork would make further unwinding increasingly difficult. The helicase would stall, and replication would halt. Topoisomerase does not itself unwind the duplex; it changes the linking number of the DNA by breaking and rejoining the backbone.

Another misconception is that topoisomerase acts at the fork itself. In reality, topoisomerase acts ahead of the fork, on the still-duplex DNA that is becoming overwound. The enzyme binds to the duplex, cuts one or both strands, passes the DNA through the break, and reseals it.

## Summary and Key Takeaways

DNA replication at the fork is a masterpiece of molecular coordination. The antiparallel nature of DNA forces the cell to synthesize one strand continuously and the other discontinuously. The lagging strand is made as Okazaki fragments, each initiated by an RNA primer, extended by DNA polymerase, and joined by ligase. The trombone model explains how the two polymerases work together despite opposite directions of synthesis.

### Essential Concepts

The replication fork is the site of active DNA synthesis, where helicase unwinds the duplex and two polymerases synthesize daughter strands. The leading strand is made continuously; the lagging strand is made in fragments. Primase provides the RNA primers required for polymerase initiation. The sliding clamp confers processivity. DNA polymerase I removes RNA primers and fills gaps. DNA ligase seals nicks. The trombone model coordinates the two polymerases.

### Quick Revision Checklist

- DNA polymerase synthesizes only in the 5′ to 3′ direction.
- The leading strand is synthesized continuously; the lagging strand is synthesized discontinuously.
- Okazaki fragments are short DNA pieces on the lagging strand, 1,000–2,000 nucleotides in bacteria and 100–200 nucleotides in eukaryotes.
- Primase (DnaG in *E. coli*) synthesizes RNA primers.
- DNA polymerase III is the replicative polymerase in bacteria; DNA polymerase I removes RNA primers.
- DNA ligase seals the nicks between Okazaki fragments.
- The trombone model describes how leading and lagging strand polymerases are coordinated.
- Topoisomerase relieves supercoiling ahead of the fork; helicase unwinds the duplex.

## Frequently Asked Questions

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

The lagging strand is synthesized in fragments because DNA polymerase can only synthesize in the 5′ to 3′ direction. On the lagging strand template, which is oriented in the 3′ to 5′ direction relative to the fork, the polymerase would have to move away from the fork to synthesize continuously. Instead, the polymerase repeatedly synthesizes short segments in the 5′ to 3′ direction, each starting from a new RNA primer, as the fork advances.

### What are Okazaki fragments?

Okazaki fragments are short segments of DNA synthesized on the lagging strand during DNA replication. In bacteria, they are typically 1,000 to 2,000 nucleotides long; in eukaryotes, they are 100 to 200 nucleotides. Each fragment begins with an RNA primer and is later joined to the adjacent fragment by DNA ligase after the primer is removed and replaced with DNA.

### What is the role of RNA primers in lagging strand synthesis?

RNA primers provide the 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 synthesizes a short RNA primer (10–12 nucleotides in bacteria) complementary to the template, and DNA polymerase extends this primer. The RNA primer is later removed and replaced with DNA.

### How are Okazaki fragments joined together?

Okazaki fragments are joined through a three-step process. First, DNA polymerase I removes the RNA primer at the 5′ end of each fragment using its 5′ to 3′ exonuclease activity. Second, the same enzyme fills the resulting gap with DNA using its polymerase activity. Third, DNA ligase seals the remaining nick by forming a phosphodiester bond between the 3′-OH of the newly synthesized DNA and the 5′-phosphate of the adjacent fragment.

### What is the trombone model?

The trombone model is a structural model of the replisome that explains how leading and lagging strand synthesis are coordinated. In this model, the two DNA polymerase molecules are physically linked, and the lagging strand template forms a loop that grows and collapses as Okazaki fragments are synthesized. The loop allows the lagging strand polymerase to synthesize in the 5′ to 3′ direction while moving with the fork.

### Why is the leading strand synthesized continuously?

The leading strand is synthesized continuously because its template is oriented such that DNA polymerase can synthesize in the 5′ to 3′ direction while moving in the same direction as the fork. The polymerase requires only a single RNA primer at the origin and then synthesizes the entire strand without dissociating.

### What happens if DNA ligase is defective?

If DNA ligase is defective, Okazaki fragments cannot be joined. The lagging strand remains fragmented, with nicks between adjacent fragments. This leads to the accumulation of single-strand breaks, which can cause [replication fork collapse](/knowledge/molecular-biology/replication-fork-collapse) and DNA damage. In humans, mutations in DNA ligase I cause a rare immunodeficiency syndrome characterized by sensitivity to DNA-damaging agents. Defects in ligase are lethal in most organisms because the lagging strand cannot be completed.

## Key Takeaways

- DNA replication is semiconservative, with each daughter molecule containing one parental and one newly synthesized strand, as established by the Meselson-Stahl experiment and described under [Semi Conservative Replication](/knowledge/molecular-biology/semi-conservative-replication).
- The replication fork is the dynamic site of DNA synthesis, where helicase unwinds the duplex and two DNA polymerases synthesize daughter strands in the 5′ to 3′ direction.
- The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously as Okazaki fragments.
- Primase synthesizes RNA primers that provide the free 3′-OH required by DNA polymerase; the leading strand needs one primer, the lagging strand needs one per fragment.
- DNA polymerase III is the replicative polymerase in bacteria, with the β sliding clamp conferring high processivity and 3′ to 5′ exonuclease activity providing proofreading.
- DNA polymerase I removes RNA primers and fills the resulting gaps, and DNA ligase seals the nicks to produce a continuous lagging strand.
- The trombone model explains how the two polymerases are coordinated through looping of the lagging strand template, allowing both to advance with the fork.
- Topoisomerase relieves positive supercoiling ahead of the fork; without it, replication stalls due to torsional stress.

## Further Reading

- Burgers PMJ, Kunkel TA. *Eukaryotic [DNA Replication Fork](/blog/guides/dna-replication-fork)*. Annual review of biochemistry. 2017. [PubMed 28301743](https://doi.org/10.1146/annurev-biochem-061516-044709)
- Canal B et al. *The DNA replication checkpoint limits Okazaki fragment accumulation to protect and restart stalled forks*. Molecular cell. 2025. [PubMed 40578347](https://doi.org/10.1016/j.molcel.2025.06.001)
- Xu Y et al. *DNA nicks in both leading and lagging strand templates can trigger break-induced replication*. Molecular cell. 2025. [PubMed 39561776](https://doi.org/10.1016/j.molcel.2024.10.026)
- Rouillon C et al. *CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands*. [Nucleic acids research](/blog/news/nucleic-acids-research). 2023. [PubMed 36942484](https://doi.org/10.1093/nar/gkad171)
- Nasheuer HP, Onwubiko NO. *Lagging Strand Initiation Processes in DNA Replication of Eukaryotes-Strings of Highly Coordinated Reactions Governed by Multiprotein Complexes*. Genes. 2023. [PubMed 37239371](https://doi.org/10.3390/genes14051012)
- He R, Zhang Z. *Rad53 arrests leading and lagging strand DNA synthesis via distinct mechanisms in response to DNA replication stress*. BioEssays : news and reviews in molecular, cellular and [developmental biology](/blog/careers/developmental-biology). 2022. [PubMed 35778827](https://doi.org/10.1002/bies.202200061)

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