GO:0033677 DNA/RNA helicase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0033677 DNA/RNA helicase activity describes the ATP-dependent unwinding of a DNA/RNA duplex, a hybrid in which one strand is DNA and the complementary strand is RNA.
• This activity is essential for resolving R-loops and DNA:RNA hybrids that arise during transcription, DNA replication, and DNA double-strand break repair.
• Key enzymes include DHX9, DHX33, DDX5, ADAR1, INTS6-SOSS1, and the yeast Rad3 and Irc3 helicases, which couple ATP hydrolysis to hybrid unwinding.
• Loss of DNA/RNA helicase activity causes R-loop accumulation, replication stress, and genome instability, contributing to cancer and other diseases.
• CRISPR knockout, point-mutation knock-in, and overexpression models enable causal testing of these helicases in R-loop biology and therapy resistance.
• EDITGENE provides end-to-end CRISPR cell model and screening services to study DNA/RNA helicase activity in disease-relevant contexts.
Description
DNA/RNA helicase activity (GO:0033677) is a molecular function that catalyzes the ATP-dependent unwinding of a DNA/RNA duplex, a double helix formed when a DNA strand pairs with a complementary RNA strand. Such DNA:RNA hybrids, often called R-loops when they involve a displaced single-stranded DNA, are pervasive intermediates of transcription and genome maintenance. The ability to resolve these hybrids is critical because persistent R-loops interfere with replication, transcription, and DNA repair, leading to genome instability. Enzymes with DNA/RNA helicase activity therefore sit at the interface of RNA metabolism and genome integrity. Researchers study GO:0033677 to understand how cells manage transcription-associated DNA:RNA hybrids and to identify therapeutic vulnerabilities in cancers that depend on these helicases. For example, DHX33 was shown to possess dual DNA/RNA helicase activity in vitro, directly linking its biochemical function to hybrid resolution. Similarly, the yeast Rad3 protein was characterized as a DNA and DNA:RNA helicase, establishing an early paradigm for this activity. More recently, the INTS6-SOSS1 complex was found to facilitate DNA:RNA hybrid autoregulation at double-strand breaks, expanding the known repertoire of hybrid-processing machines. Because DNA/RNA hybrids are central to replication stress responses and DNA repair, defects in helicases that unwind them can sensitize cells to chemotherapeutic agents such as cisplatin. This makes GO:0033677 a high-value target for mechanistic studies and for the development of CRISPR-based disease models. The sections below synthesize the definition, mechanism, key genes, disease links, and experimental strategies for investigating this activity.
DNA/RNA helicase activity At A Glance
| GO ID | GO:0033677 |
|---|---|
| GO term | DNA/RNA helicase activity |
| Ontology | molecular_function |
| Synonym | ATP-dependent DNA/RNA helicase activity |
| Definition | Unwinding of a DNA/RNA duplex, i.e. a double helix in which a strand of DNA pairs with a complementary strand of RNA, driven by ATP hydrolysis. |
| Major function | ATP-dependent separation of DNA-RNA hybrids, including R-loops and transcription/replication intermediates. |
| Representative enzymes | DHX9, DHX33, DDX5, ADAR1, INTS6-SOSS1, Rad3, Irc3. |
| Biological context | Transcription, DNA replication, DNA double-strand break repair, and R-loop homeostasis. |
| Disease relevance | Cancer, replication stress, chemoresistance, and genome instability syndromes. |
What Is GO:0033677?
GO:0033677 DNA/RNA helicase activity is defined as the unwinding of a DNA/RNA duplex, i.e., a double helix in which a strand of DNA pairs with a complementary strand of RNA, driven by ATP hydrolysis. In other words, it is an ATP-dependent motor activity that separates a DNA-RNA hybrid into single strands. This activity is distinct from DNA-DNA or RNA-RNA helicase activities because its substrate is a mixed DNA/RNA duplex. The official synonym is ATP-dependent DNA/RNA helicase activity.
Why Is DNA/RNA helicase activity Important in Cell Biology?
DNA/RNA helicase activity is important because DNA:RNA hybrids are unavoidable byproducts of transcription and are particularly abundant at sites of replication stress and DNA damage. If these hybrids are not resolved, they can block replication forks, cause transcription-replication conflicts, and trigger DNA double-strand breaks. Helicases with this activity, such as DHX9 and DDX5, are recruited to chromatin to resolve R-loops and promote repair. Consequently, loss of DNA/RNA helicase activity leads to genome instability and can sensitize cancer cells to DNA-damaging agents like cisplatin. Understanding this activity is therefore central to cancer biology, neurobiology, and the development of targeted therapies.
• Maintains R-loop homeostasis by unwinding DNA:RNA hybrids that accumulate during transcription.
• Supports DNA double-strand break repair by resolving DNA:RNA hybrids at damage sites.
• Prevents replication stress and transcription-replication conflicts.
• Modulates sensitivity to chemotherapeutic agents such as cisplatin in ovarian cancer.
• Involved in chromatin recruitment of helicases like DHX9 via TDRD3.
• Links RNA editing and ATR activation through ADAR1 in replication stress responses.
• Provides a biochemical basis for dual DNA/RNA helicase enzymes such as DHX33.
• Conserved from yeast to humans, as shown for Rad3 and Irc3 helicases.
• Potential target for cancer therapy in tumors with high R-loop burden.
• Enables CRISPR-based functional genomics of hybrid resolution pathways.
What Happens During DNA/RNA helicase activity?
Substrate recognition and binding
In simple terms: The helicase first finds and grabs the DNA-RNA hybrid.
DNA/RNA helicases must recognize a DNA:RNA duplex among the many nucleic acid structures in the cell. For example, recombinant DHX33 was shown to bind and unwind DNA/RNA hybrids in an ATP-dependent manner. Similarly, the yeast Rad3 protein was characterized as a DNA and DNA:RNA helicase, indicating that it can engage mixed duplexes. The INTS6-SOSS1 complex is recruited to DNA double-strand breaks where it facilitates DNA:RNA hybrid autoregulation, suggesting that substrate recognition is coupled to damage signaling.
ATP hydrolysis and strand separation
In simple terms: The helicase burns ATP to pull the two strands apart.
Once bound, the helicase couples ATP hydrolysis to the unwinding of the DNA/RNA duplex. The mitochondrial Irc3 helicase of Ogataea polymorpha displays dual DNA- and RNA-stimulated ATPase activity, demonstrating that ATP turnover is stimulated by both DNA and RNA. DHX33 possesses dual DNA/RNA helicase activity, confirming that ATP hydrolysis drives separation of the hybrid. This step is essential for converting a stable DNA:RNA hybrid into single-stranded DNA and RNA, which can then be further processed by other factors.
R-loop resolution and hybrid clearance
In simple terms: The helicase removes R-loops that form when RNA sticks to DNA.
R-loops are DNA:RNA hybrids that leave a displaced single-stranded DNA. TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution, directly linking a DNA/RNA helicase to hybrid clearance. BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair. ADAR1 links R-loop homeostasis to ATR activation in replication stress responses, showing that hybrid clearance is integrated with checkpoint signaling. These examples illustrate that DNA/RNA helicase activity is a key step in R-loop resolution.
Coupling to DNA repair and replication stress responses
In simple terms: After unwinding, the cell uses the freed strands to repair DNA or pause replication safely.
DNA/RNA helicase activity is often coupled to downstream repair and signaling. The INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks, indicating a role in break repair. DDX5, promoted by BRCA2, resolves DNA-RNA hybrids at DNA breaks to facilitate their repair. ADAR1 links R-loop homeostasis to ATR activation, which is a central replication stress response kinase. In ovarian cancer cells, targeting SLFN11 and ATR can overcome cisplatin resistance, highlighting the therapeutic relevance of these pathways.
Key Genes Involved in GO:0033677 DNA/RNA helicase activity
The following genes encode proteins with demonstrated or putative DNA/RNA helicase activity (GO:0033677) or are directly involved in DNA:RNA hybrid resolution.
| Gene | Major Role | Research Relevance |
|---|---|---|
| DHX33 | Dual DNA/RNA helicase that unwinds DNA/RNA hybrids in an ATP-dependent manner. | Biochemical studies of helicase activity; cancer cell proliferation. |
| DHX9 | Recruited to chromatin by TDRD3 to resolve R-loops. | R-loop biology, transcription-replication conflicts, cancer. |
| DDX5 | Promoted by BRCA2 to resolve DNA-RNA hybrids at DNA breaks. | DNA repair, breast/ovarian cancer, hybrid resolution. |
| ADAR1 | Links R-loop homeostasis to ATR activation in replication stress. | Replication stress, innate immunity, cancer. |
| INTS6 | Part of INTS6-SOSS1 complex that facilitates DNA:RNA hybrid autoregulation at double-strand breaks. | DNA damage response, hybrid regulation. |
| SOSS1 | Complex with INTS6 for DNA:RNA hybrid autoregulation at DSBs. | DSB repair, genome stability. |
| BRCA2 | Promotes DDX5-mediated DNA-RNA hybrid resolution at DNA breaks. | Homologous recombination, cancer predisposition. |
| TDRD3 | Promotes DHX9 chromatin recruitment and R-loop resolution. | R-loop resolution, chromatin biology. |
| SLFN11 | Target for overcoming cisplatin resistance; linked to replication stress. | Chemoresistance, ovarian cancer. |
| ATR | Kinase activated by R-loop homeostasis via ADAR1; target in cisplatin resistance. | Replication stress response, cancer therapy. |
| Rad3 (yeast) | DNA and DNA:RNA helicase with putative RNA helicase activity. | Model organism studies of helicase function. |
| Irc3 (yeast) | Mitochondrial helicase with dual DNA- and RNA-stimulated ATPase activity. | Mitochondrial RNA/DNA metabolism. |
| DDX5 (alias p68) | RNA helicase with DNA-RNA hybrid resolution activity at breaks. | Cancer biology, DNA repair. |
| DHX9 (alias RHA) | Helicase that resolves R-loops upon TDRD3 recruitment. | Transcription, genome stability. |
| ADAR1 (alias DRADA) | RNA editing enzyme that also influences R-loop homeostasis. | Replication stress, interferon response. |
| INTS6 (alias DDX26) | Component of Integrator complex and INTS6-SOSS1 hybrid regulator. | DSB repair, RNA processing. |
How Is DNA/RNA helicase activity Regulated?
DNA/RNA helicase activity is regulated at multiple levels. Recruitment to sites of DNA damage or replication stress is a key control point: TDRD3 promotes DHX9 chromatin recruitment for R-loop resolution, and BRCA2 promotes DDX5 localization to DNA breaks. ADAR1 links R-loop homeostasis to ATR activation, meaning that the replication stress kinase ATR can be activated by changes in hybrid levels. The INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks, suggesting a feedback mechanism. Additionally, the ATPase activity of Irc3 is stimulated by both DNA and RNA, indicating substrate-level regulation. These layers ensure that DNA/RNA helicase activity is deployed when and where it is needed.
DNA/RNA helicase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| SLFN11 | Cisplatin resistance in ovarian cancer | Knockout in ovarian cancer cell lines; drug sensitivity assays. |
| ATR | Replication stress response and cisplatin resistance | Point mutation or knockout to test ATR inhibitor sensitivity. |
| BRCA2 | Breast/ovarian cancer, DNA repair | Knock-in of patient mutations; hybrid resolution assays. |
| DDX5 | DNA repair and cancer | Knockout and overexpression in cancer cells; R-loop detection. |
| DHX9 | R-loop resolution and genome stability | Knockout with TDRD3 rescue; chromatin recruitment studies. |
Cancer and chemoresistance
DNA/RNA helicase activity is frequently dysregulated in cancer. In ovarian cancer cells, targeting SLFN11 and ATR can overcome cisplatin resistance, and this is linked to replication stress and R-loop biology. BRCA2, which promotes DDX5-mediated hybrid resolution, is a well-known cancer predisposition gene. Loss of DDX5 function at DNA breaks impairs repair and can contribute to genomic instability. Therefore, inhibitors of DNA/RNA helicases or their regulators are being explored as anticancer strategies.
Replication stress and genome instability
Defects in DNA/RNA helicase activity lead to R-loop accumulation, which causes replication stress and DNA damage. ADAR1 links R-loop homeostasis to ATR activation, and loss of this regulation can exacerbate replication stress. DHX9, when not properly recruited by TDRD3, fails to resolve R-loops, leading to transcription-replication conflicts. The INTS6-SOSS1 complex is important for hybrid autoregulation at double-strand breaks, and its dysfunction may contribute to genome instability. These mechanisms are relevant to cancer and potentially to developmental disorders.
Mitochondrial dysfunction
The mitochondrial Irc3 helicase of Ogataea polymorpha displays dual DNA- and RNA-stimulated ATPase activity, suggesting a role in mitochondrial nucleic acid metabolism. While direct human disease links are not established in the provided literature, mitochondrial helicases are essential for mitochondrial genome maintenance. This highlights the broader importance of DNA/RNA helicase activity beyond the nucleus.
From DNA/RNA helicase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of DHX33 reduce DNA/RNA helicase activity? | CRISPR knockout of DHX33 in cancer cell lines followed by biochemical helicase assays. |
| How does TDRD3-mediated recruitment of DHX9 affect R-loop resolution? | Knockout of TDRD3 or DHX9, or tagged knock-in of DHX9, with R-loop imaging. |
| What is the role of ADAR1 in ATR activation during replication stress? | Point mutation of ADAR1 editing domain or knockout, combined with ATR signaling readouts. |
| Can INTS6-SOSS1 complex formation be disrupted to study DSB repair? | Knockout of INTS6 or SOSS1 subunits, or knock-in of tagged versions, with DSB repair assays. |
| Does BRCA2 promote DDX5-mediated hybrid resolution at breaks? | Knock-in of BRCA2 mutants, DDX5 knockout, and DNA break assays. |
| Is Irc3 ATPase activity stimulated by DNA and RNA? | Overexpression of wild-type and mutant Irc3 in yeast, followed by ATPase assays. |
How to Study the DNA/RNA helicase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Helicase assay with DNA/RNA duplex | ATP-dependent unwinding of DNA/RNA hybrids | Confirming direct DNA/RNA helicase activity of DHX33 or Rad3. |
| ATPase assay | ATP hydrolysis stimulated by DNA or RNA | Characterizing Irc3 dual-stimulated ATPase activity. |
| R-loop detection (S9.6 antibody) | Levels and localization of DNA:RNA hybrids | Assessing R-loop resolution by DHX9/TDRD3. |
| Chromatin immunoprecipitation (ChIP) | Recruitment of helicases to chromatin | Studying TDRD3-dependent DHX9 recruitment. |
| DNA double-strand break repair assays | Efficiency of break repair | Testing INTS6-SOSS1 and BRCA2-DDX5 functions. |
| CRISPR knockout screening | Gene essentiality and drug sensitivity | Identifying helicases required for cisplatin resistance. |
| Co-immunoprecipitation | Protein-protein interactions | Detecting INTS6-SOSS1 complex formation. |
| RNA-seq / Ribo-seq | Transcriptome and translatome changes | Global effects of helicase loss on gene expression. |
Biochemical helicase assays
Direct measurement of DNA/RNA helicase activity uses synthetic DNA/RNA duplex substrates and monitors strand separation in the presence of ATP. Recombinant DHX33 was shown to possess dual DNA/RNA helicase activity using such assays. Similarly, the Rad3 protein was characterized as a DNA and DNA:RNA helicase using biochemical fractionation and helicase assays. These methods are essential to confirm that a candidate enzyme directly unwinds DNA/RNA hybrids.
R-loop detection and imaging
R-loops can be detected using the S9.6 antibody or by native gel electrophoresis. TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution, which was demonstrated using R-loop detection methods. ADAR1 links R-loop homeostasis to ATR activation, and R-loop levels were monitored in replication stress conditions. These techniques allow researchers to quantify hybrid accumulation upon loss or gain of helicase function.
Genome-wide and proteomic approaches
Proteomics and genome-wide screens can identify interactors and targets of DNA/RNA helicases. The INTS6-SOSS1 complex was identified and characterized using biochemical purification and functional assays at double-strand breaks. BRCA2 and DDX5 interactions were studied using co-immunoprecipitation and repair assays. CRISPR library screening can systematically test which helicases are required for hybrid resolution and cell survival.
CRISPR-based functional genomics
CRISPR knockout and knock-in models enable causal testing of DNA/RNA helicase genes. For example, targeting SLFN11 and ATR with CRISPR can reveal their roles in cisplatin resistance. Knockout of DHX9 or TDRD3 can be used to study R-loop resolution. These approaches are powerful for linking genotype to phenotype in disease-relevant cell models.
How CRISPR Can Be Used to Study GO:0033677 DNA/RNA helicase activity
Knockout
CRISPR knockout is used to eliminate DNA/RNA helicase genes and assess loss-of-function phenotypes. For example, knocking out DHX9 or TDRD3 can reveal defects in R-loop resolution. Knockout of SLFN11 or ATR can test their roles in cisplatin resistance. These models are essential for determining whether a helicase is required for hybrid clearance and genome stability.
Point Mutation
Point mutations can dissect catalytic residues or regulatory domains. For instance, mutating the ATPase domain of a helicase can abolish DNA/RNA helicase activity while preserving protein interactions. Point mutations in ADAR1 can separate its editing function from its role in R-loop homeostasis. Such models provide mechanistic insights beyond simple knockout.
Knock-in
Knock-in of tagged or mutant versions of helicases allows tracking and functional studies. A tagged knock-in of DHX9 can be used to study its chromatin recruitment by TDRD3. Knock-in of patient-derived BRCA2 mutations can test their impact on DDX5-mediated hybrid resolution. These models are valuable for studying disease-associated variants.
Overexpression
Overexpression of wild-type or mutant helicases can test gain-of-function effects. Overexpression of DHX33 confirmed its dual DNA/RNA helicase activity in vitro. Overexpression of Irc3 in yeast was used to study its ATPase activity. Overexpression can also sensitize cells to DNA-damaging agents, revealing therapeutic opportunities.
How EDITGENE Supports DNA/RNA helicase activity Research
Researchers studying DNA/RNA helicase activity-related genes often need to determine whether a candidate gene is causally involved in hybrid resolution, replication stress, or drug resistance. This requires precise genetic models that can isolate the contribution of a single gene or mutation. EDITGENE provides a comprehensive suite of CRISPR services to generate such models efficiently.
Contact EDITGENE today to design your custom CRISPR model for DNA/RNA helicase activity research.
Frequently Asked Questions About DNA/RNA helicase activity
What is DNA/RNA helicase activity?
DNA/RNA helicase activity (GO:0033677) is the ATP-dependent unwinding of a DNA/RNA duplex, a double helix in which a DNA strand pairs with a complementary RNA strand.
What genes are involved in DNA/RNA helicase activity?
Key genes include DHX33, DHX9, DDX5, ADAR1, INTS6, SOSS1, BRCA2, TDRD3, and the yeast Rad3 and Irc3 helicases.
What is the GO ID for DNA/RNA helicase activity?
The GO ID is GO:0033677.
How does DNA/RNA helicase activity relate to R-loops?
R-loops are DNA:RNA hybrids that can be resolved by helicases such as DHX9 and DDX5, which unwind the hybrid to maintain genome stability.
Which diseases are linked to DNA/RNA helicase activity?
Dysregulation is linked to cancer, chemoresistance, replication stress, and genome instability.
What methods are used to study DNA/RNA helicase activity?
Common methods include biochemical helicase assays, ATPase assays, R-loop detection, ChIP, and CRISPR screens.
Can CRISPR be used to study DNA/RNA helicase activity?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models are widely used to dissect gene function in hybrid resolution.
What is the role of DHX33 in DNA/RNA helicase activity?
Recombinant DHX33 possesses dual DNA/RNA helicase activity, directly unwinding DNA/RNA hybrids in an ATP-dependent manner.
How does ADAR1 regulate R-loop homeostasis?
ADAR1 links R-loop homeostasis to ATR activation during replication stress, integrating hybrid metabolism with checkpoint signaling.
What is the clinical relevance of DNA/RNA helicase activity?
It is relevant to cancer therapy, as targeting helicases or related pathways can overcome cisplatin resistance in ovarian cancer cells.
Conclusion
DNA/RNA helicase activity (GO:0033677) is a fundamental molecular function that resolves DNA:RNA hybrids, protecting genome integrity during transcription, replication, and repair. Its dysregulation contributes to cancer and chemoresistance, making it a promising therapeutic target. Advances in CRISPR-based models and biochemical assays continue to illuminate the mechanisms and disease relevance of this activity. EDITGENE offers comprehensive services to support research on DNA/RNA helicase activity and its associated genes.
References
- 1. König P et al.. 2024. SLFN11 and ATR as targets for overcoming cisplatin resistance in ovarian cancer cells.. Biochim Biophys Acta Mol Basis Dis 1870(8):167448 PMID: 39117290
- 2. Wang X et al.. 2019. Recombinant DHX33 Protein Possesses Dual DNA/RNA Helicase Activity.. Biochemistry 58(4):250-258 PMID: 29870660
- 3. Zhang B et al.. 2023. ADAR1 links R-loop homeostasis to ATR activation in replication stress response.. Nucleic Acids Res 51(21):11668-11687 PMID: 37831098
- 4. Yuan W et al.. 2021. TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution.. Nucleic Acids Res 49(15):8573-8591 PMID: 34329467
- 5. Long Q et al.. 2024. Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks.. Nucleic Acids Res 52(21):13036-13056 PMID: 39445827
- 6. Piljukov VJ et al.. 2023. Mitochondrial Irc3 helicase of the thermotolerant yeast Ogataea polymorpha displays dual DNA- and RNA-stimulated ATPase activity.. Mitochondrion 69:130-139 PMID: 36764503
- 7. Deschavanne PJ et al.. 1993. The Rad3 protein from Saccharomyces cerevisiae: a DNA and DNA:RNA helicase with putative RNA helicase activity.. Mol Microbiol 7(6):831-5 PMID: 8387143
- 8. Sessa G et al.. 2021. BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.. EMBO J 40(7):e106018 PMID: 33634895
