GO:0043137 DNA replication, removal of RNA primer: Okazaki Maturation Pathway, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0043137 (DNA replication, removal of RNA primer) describes the removal of the Okazaki RNA primer from the lagging strand of replicating DNA by the combined action of DNA polymerase, DNA helicase and an endonuclease.
• The process is also called Okazaki initiator RNA removal and is essential for converting discontinuous Okazaki fragments into a continuous lagging strand.
• Two major, non-exclusive routes have been visualized in eukaryotic cells: a flap cleavage pathway and an exonucleolytic pathway.
• Key enzymes include FEN1, DNA2, RNase H2, PCNA, Pol delta and the helicase/nuclease Dna2, whose coordinated action removes RNA-DNA hybrids.
• In organelles, dedicated factors such as PEN1 in maize plastids and mammalian mitochondrial priming/removal machinery carry out analogous RNA primer removal.
• Dysregulation of primer removal is linked to genome instability, mitochondrial disease and cancer, making it a target for mechanistic and therapeutic studies.
Description
DNA replication, removal of RNA primer (GO:0043137) is the biological process that eliminates the short RNA oligonucleotide used to initiate each Okazaki fragment on the lagging strand, allowing DNA polymerase to fill the gap and DNA ligase to seal the nick. Without this step, lagging-strand synthesis would remain fragmented and the genome would accumulate persistent RNA-DNA hybrids. The term is defined in QuickGO as the removal of the Okazaki RNA primer from the lagging strand of replicating DNA, by a combination of the actions of DNA polymerase, DNA helicase and an endonuclease, and its synonym is Okazaki initiator RNA removal. Researchers study GO:0043137 because it sits at the intersection of replication, DNA repair and genome stability, and because defects in primer removal are associated with human disease, including mitochondrial disorders and cancer. Recent single-molecule tracking has begun to resolve how the relevant enzymes find and process RNA-DNA hybrids in real time, while genetic studies in plants have identified dedicated plastid factors such as PEN1. In eukaryotic nuclei, direct visualization of Okazaki fragment intermediates has provided support for both flap cleavage and exonucleolytic pathways. This article summarizes the authoritative definition, the molecular players, the disease connections and the experimental models used to investigate GO:0043137.
DNA replication, removal of RNA primer At A Glance
| GO ID | GO:0043137 |
|---|---|
| GO term | DNA replication, removal of RNA primer |
| Ontology | biological_process |
| Synonym | Okazaki initiator RNA removal |
| Major function | Removal of the Okazaki RNA primer from the lagging strand of replicating DNA by DNA polymerase, DNA helicase and an endonuclease |
| Related processes | Okazaki fragment metabolism, lagging-strand DNA synthesis, DNA repair |
| Key enzymatic activities | Flap endonuclease, 5'-3' exonuclease, helicase, DNA polymerase |
| Cellular contexts | Nuclear DNA replication, mitochondrial DNA replication, plastid DNA replication |
| Representative factors | FEN1, DNA2, RNase H2, PCNA, Pol delta, PEN1 |
What Is GO:0043137?
GO:0043137 is the biological process in which the RNA primer of an Okazaki fragment is removed from the lagging strand during DNA replication. According to the QuickGO definition, this removal is achieved by a combination of DNA polymerase, DNA helicase and an endonuclease activities. The process is synonymous with Okazaki initiator RNA removal and is a required maturation step that converts short, RNA-primed Okazaki fragments into ligatable DNA.
Why Is DNA replication, removal of RNA primer Important in Cell Biology?
Removal of the RNA primer is essential for faithful duplication of the genome because unrepaired RNA-DNA hybrids block ligation and can cause replication fork stalling, strand breaks and mutagenesis. The process is conserved across eukaryotes, bacteria and organelles, and its dysfunction has been linked to mitochondrial disease and cancer. Understanding GO:0043137 therefore informs basic replication biology and provides a mechanistic basis for interpreting disease-associated mutations in primer-removal enzymes.
• Ensures continuous lagging-strand synthesis by converting RNA-primed Okazaki fragments into ligatable DNA.
• Prevents persistent RNA-DNA hybrids that can trigger genome instability.
• Provides a model for coordinated polymerase, helicase and nuclease action.
• Is conserved in mitochondria and plastids, where dedicated factors such as PEN1 operate.
• Links replication mechanics to human mitochondrial disease.
• Contributes to cancer biology through genome maintenance pathways.
• Offers targets for single-molecule imaging of enzyme dynamics.
• Supports comparative studies of bacteriophage and eukaryotic primer removal.
• Informs CRISPR screens for replication-stress vulnerabilities.
• Guides interpretation of variants in FEN1, DNA2 and RNase H2.
What Happens During DNA replication, removal of RNA primer?
Initiation and RNA priming of Okazaki fragments
In simple terms: The lagging strand starts each fragment with a short RNA piece.
During lagging-strand synthesis, DNA polymerase alpha primase lays down a short RNA primer that is extended by DNA polymerase to form an Okazaki fragment. This RNA segment must later be removed to allow complete DNA synthesis.
Strand displacement and flap formation
In simple terms: The next fragment pushes the RNA aside, creating a flap.
As the downstream Okazaki fragment is synthesized, the polymerase displaces the 5' end of the upstream RNA primer, generating a flap structure that becomes the substrate for nucleases. This strand-displacement step is a key feature of the flap cleavage pathway.
Flap cleavage by FEN1 and DNA2
In simple terms: Special scissors cut off the RNA flap.
Flap endonuclease 1 (FEN1) cleaves the displaced RNA-DNA flap, while the helicase/nuclease DNA2 can process longer flaps in a coordinated manner. Direct visualization of Okazaki fragment intermediates supports the involvement of both flap cleavage and exonucleolytic routes.
Exonucleolytic degradation of RNA primers
In simple terms: Other enzymes chew the RNA away base by base.
In addition to flap cleavage, exonucleolytic pathways degrade the RNA primer, and RNase H2 can remove ribonucleotides embedded in DNA. Single-molecule tracking of RNA-DNA hybrid removal enzymes has revealed dynamic behavior consistent with these redundant mechanisms.
Gap filling and ligation
In simple terms: The remaining DNA gap is filled and sealed.
After primer removal, DNA polymerase delta fills the gap and DNA ligase I seals the nick, completing Okazaki fragment maturation. This final step depends on prior removal of the RNA primer and is essential for genome integrity.
Organellar and non-nuclear variants
In simple terms: Mitochondria and plastids use their own primer-removal factors.
In mammalian mitochondria, primer removal during DNA replication involves distinct factors and is important for mitochondrial genome maintenance. In maize plastids, PEN1 catalyses RNA primer removal, and the mechanism has been highlighted as a conserved theme in organellar replication.
Key Genes Involved in GO:0043137 DNA replication, removal of RNA primer
The following genes and proteins are central to RNA primer removal during DNA replication, based on the cited literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| FEN1 | Flap endonuclease that cleaves RNA-DNA flaps during Okazaki maturation | Core enzyme for flap cleavage pathway studies |
| DNA2 | Helicase/nuclease that processes long flaps and coordinates with FEN1 | Target for mechanistic and single-molecule studies |
| RNase H2 | Removes ribonucleotides from RNA-DNA hybrids | Linked to genome stability and hybrid removal |
| PCNA | Sliding clamp that coordinates primer-removal enzymes | Platform for assembly and regulation studies |
| POLA1 | Primase subunit that synthesizes the RNA primer | Upstream factor defining the substrate for removal |
| POLD1 | DNA polymerase delta that fills gaps after primer removal | Effector of lagging-strand completion |
| LIG1 | DNA ligase I that seals nicks after primer removal | Readout of successful maturation |
| PEN1 | Plastid factor that catalyses RNA primer removal in maize | Plant organellar model for primer removal |
| POLG | Mitochondrial DNA polymerase involved in mitochondrial replication | Mitochondrial disease model |
| T7 gene 6 exonuclease | Bacteriophage flap endonuclease with roles in RNA primer removal | Comparative model for primer removal |
| RPA | Single-stranded DNA-binding protein that facilitates flap processing | Accessory factor in reconstitution assays |
| Dna2-FEN1 axis | Coordinated nuclease activities for flap removal | Pathway dissection and inhibitor studies |
| RNase H1 | Processes RNA-DNA hybrids in mitochondria and nuclei | Organellar hybrid removal studies |
| MCM helicase | Unwinds DNA to expose primer-removal substrates | Replication fork studies |
| CTF4/AND-1 | Coordinates lagging-strand factors | Assembly and regulation studies |
| TopBP1 | Replication stress response factor | Links primer removal to checkpoint signaling |
| TIMELESS | Fork protection factor | Replication stress models |
How Is DNA replication, removal of RNA primer Regulated?
RNA primer removal is regulated by protein-protein interactions and post-translational modifications that coordinate FEN1, DNA2, PCNA and polymerase delta at the replication fork. Single-molecule studies show that the enzymes dynamically associate with RNA-DNA hybrids, suggesting that their residence times and handoff are regulated. In mitochondria, primer removal is coupled to the distinct replication machinery and is influenced by factors that maintain the mitochondrial genome. In plastids, PEN1 provides a dedicated regulatory node for primer removal.
DNA replication, removal of RNA primer and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| FEN1 | Genome instability and cancer predisposition | Knockout and point-mutation cell lines |
| DNA2 | Replication stress and mitochondrial dysfunction | Knockout and overexpression models |
| RNase H2 | RNA-DNA hybrid accumulation and genome instability | Knock-in of patient variants |
| PEN1 | Plastid DNA replication defects in maize | Plant knockout and complementation |
| POLG | Mitochondrial disease | Mitochondrial disease cell models |
Mitochondrial disease
Defects in primer removal during mammalian mitochondrial DNA replication are associated with mitochondrial dysfunction and disease phenotypes. The specialized machinery that removes RNA primers in mitochondria is therefore a focus for understanding mitochondrial genome instability.
Cancer and genome instability
Failure to remove RNA primers can lead to persistent RNA-DNA hybrids, replication stress and genome instability, processes linked to cancer development. Enzymes such as FEN1 and DNA2 are studied as potential targets in replication-stress-based therapies.
Organellar replication disorders
In plants, PEN1-mediated primer removal in plastids is essential for plastid DNA replication, and its disruption affects organellar genome maintenance. This provides a model for understanding organelle-specific replication disorders.
From DNA replication, removal of RNA primer-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is FEN1 required for Okazaki fragment maturation? | FEN1 knockout cell line |
| Does a disease variant impair primer removal? | Point-mutation knock-in |
| Can a tagged enzyme track primer removal in live cells? | Tagged knock-in |
| Does overexpression of DNA2 rescue primer-removal defects? | Overexpression cell model |
| What is the role of PEN1 in plastid replication? | Plant knockout and complementation |
| How do mitochondrial factors remove RNA primers? | Mitochondrial disease cell models |
How to Study the DNA replication, removal of RNA primer Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Single-molecule tracking | Enzyme dynamics on RNA-DNA hybrids | Real-time primer removal studies |
| In vitro reconstitution | Minimal protein requirements | Pathway dissection |
| Knockout complementation | Gene necessity | Causal gene testing |
| Fluorescence imaging | Okazaki fragment intermediates | Visualization of primer removal |
| Mitochondrial replication assay | Primer removal in organelles | Mitochondrial disease studies |
| Plastid genetics | PEN1 function in plants | Organellar replication models |
| Bacteriophage assays | T7 exonuclease roles | Comparative primer removal |
Single-molecule imaging
Single-molecule tracking of RNA-DNA hybrid removal enzymes allows real-time observation of their dynamics during lagging-strand replication. This approach can resolve how FEN1, DNA2 and related factors engage their substrates.
Biochemical reconstitution
Reconstitution assays with purified FEN1, DNA2, PCNA and polymerase delta can define the minimal requirements for primer removal. Direct visualization of Okazaki fragment intermediates supports the coexistence of flap cleavage and exonucleolytic pathways.
Genetic knockout and complementation
Knockout of candidate genes followed by complementation tests causality in primer removal. In maize, PEN1 mutants reveal the role of this factor in plastid DNA replication.
Organellar replication assays
Mitochondrial and plastid systems provide complementary models for primer removal. Mammalian mitochondrial primer removal can be studied with dedicated replication assays.
How CRISPR Can Be Used to Study GO:0043137 DNA replication, removal of RNA primer
Knockout
CRISPR knockout of FEN1, DNA2 or RNase H2 can test their requirement for RNA primer removal and Okazaki fragment maturation. Loss-of-function models reveal whether a gene is essential for lagging-strand completion.
Point Mutation
Point-mutation knock-in of disease-associated variants in primer-removal genes allows assessment of their impact on enzyme activity and genome stability. Such models can distinguish catalytic from structural functions.
Knock-in
Tagged knock-in of FEN1 or DNA2 enables live-cell imaging of primer removal dynamics. Endogenous tagging preserves physiological regulation while allowing single-molecule tracking.
Overexpression
Overexpression of primer-removal factors can test whether increased activity suppresses replication stress or alters Okazaki maturation. This approach is useful for rescue experiments.
How EDITGENE Supports DNA replication, removal of RNA primer Research
Researchers studying DNA replication, removal of RNA primer-related genes often need to determine whether a candidate gene is causally involved in primer removal, genome stability or disease. EDITGENE provides CRISPR-based cell models and screening services to test these hypotheses rigorously.
Contact EDITGENE today to design your custom CRISPR model for DNA replication, removal of RNA primer research.
Frequently Asked Questions About DNA replication, removal of RNA primer
What is GO:0043137?
GO:0043137 is the biological process DNA replication, removal of RNA primer, which removes the Okazaki RNA primer from the lagging strand by DNA polymerase, DNA helicase and an endonuclease.
What genes are involved in DNA replication, removal of RNA primer?
Key genes include FEN1, DNA2, RNase H2, PCNA, POLA1, POLD1, LIG1 and, in plastids, PEN1.
Why is RNA primer removal important?
It allows Okazaki fragments to be ligated into a continuous lagging strand and prevents genome instability from persistent RNA-DNA hybrids.
What is the synonym for GO:0043137?
The synonym is Okazaki initiator RNA removal.
Which enzymes remove the RNA primer?
Flap endonuclease FEN1, the helicase/nuclease DNA2, RNase H2 and exonucleolytic activities remove the RNA primer.
How is RNA primer removal studied?
It is studied by single-molecule tracking, biochemical reconstitution, knockout complementation and organellar replication assays.
Is RNA primer removal conserved in mitochondria?
Yes, mammalian mitochondrial DNA replication has dedicated primer-removal mechanisms.
What happens if RNA primer removal fails?
Failure leads to persistent RNA-DNA hybrids, replication stress and genome instability.
What is the role of PEN1 in primer removal?
PEN1 catalyses RNA primer removal during plastid DNA replication in maize.
How can CRISPR help study GO:0043137?
CRISPR knockout, point-mutation, knock-in and overexpression models can test the function of primer-removal genes.
Conclusion
GO:0043137, DNA replication, removal of RNA primer, is a conserved and essential step in lagging-strand DNA synthesis that converts RNA-primed Okazaki fragments into ligatable DNA. The process relies on coordinated polymerase, helicase and nuclease activities, with FEN1, DNA2 and RNase H2 as central players. Its dysfunction is linked to genome instability, mitochondrial disease and cancer, making it a compelling area for mechanistic and translational research. Advances in single-molecule imaging and organellar genetics continue to refine our understanding of how RNA primers are removed.
References
- 1. Foust DJ et al.. 2025. Single-molecule tracking of RNA-DNA hybrid removal enzymes important for lagging-strand replication.. bioRxiv PMID: 41446255
- 2. Huang X et al.. 2025. PEN1 catalyses RNA primer removal during plastid DNA replication in maize.. Nat Plants 11(7):1325-1338 PMID: 40562815
- 3. Unknown. 2025. Mechanism of RNA primer removal in plastid DNA replication.. Nat Plants 11(7):1233-1234 PMID: 40588579
- 4. Foust DJ et al.. 2026. Single-molecule tracking of RNA-DNA hybrid removal enzymes important for lagging-strand replication.. Biophys J 125(14):3718-3732 PMID: 42286923
- 5. Uhler JP et al.. 2015. Primer removal during mammalian mitochondrial DNA replication.. DNA Repair (Amst) 34:28-38 PMID: 26303841
- 6. Liu B et al.. 2017. Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells.. J Biol Chem 292(12):4777-4788 PMID: 28159842
- 7. Balakrishnan L et al.. 2013. Okazaki fragment metabolism.. Cold Spring Harb Perspect Biol 5(2) PMID: 23378587
- 8. Mitsunobu H et al.. 2014. Flap endonuclease of bacteriophage T7: Possible roles in RNA primer removal, recombination and host DNA breakdown.. Bacteriophage 4:e28507 PMID: 25105057
