GO:0097505 Rad6-Rad18 complex: Components, Assembly and Research Methods, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0097505 (Rad6-Rad18 complex) is a ubiquitin ligase complex that mediates post-replicative bypass of UV-damaged DNA and UV mutagenesis.
• The complex is a heterodimer of the ubiquitin-conjugating enzyme Rad6 and the RING-finger DNA-binding protein Rad18.
• Rad6-Rad18 binds single-stranded DNA and has single-stranded DNA-dependent ATPase activity.
• The complex monoubiquitinates PCNA to promote error-free and error-prone DNA damage tolerance.
• Rad6-Rad18 also ubiquitinates the 9-1-1 checkpoint clamp, acting as a eukaryotic SOS response.
• Dysregulation of Rad6-Rad18 is linked to cancer and chemoresistance, making it a target for CRISPR knockout and point-mutation studies.
Description
The Rad6-Rad18 complex (GO:0097505) is a conserved ubiquitin ligase complex that plays a central role in post-replicative bypass of UV-damaged DNA and UV mutagenesis. In Saccharomyces cerevisiae, the complex consists of the ubiquitin-conjugating enzyme Rad6 and Rad18, a protein containing a RING finger motif and a nucleotide binding motif. The yeast Rad6-Rad18 heterodimer has ubiquitin conjugating activity, binds single-stranded DNA, and possesses single-stranded DNA-dependent ATPase activity. This complex is essential for DNA damage tolerance and mutagenesis, and its function is conserved in higher eukaryotes. Researchers study GO:0097505 to understand how cells tolerate replication-blocking lesions and how this process contributes to genome stability and cancer.
Rad6-Rad18 complex At A Glance
| GO ID | GO:0097505 |
|---|---|
| GO term | Rad6-Rad18 complex |
| Ontology | cellular_component |
| Synonym | None |
| Major function | Ubiquitin ligase complex involved in post-replicative bypass of UV-damaged DNA and UV mutagenesis |
| Subunits | Rad6 (ubiquitin-conjugating enzyme) and Rad18 (RING finger and nucleotide binding protein) |
| Enzymatic activities | Ubiquitin conjugating activity, single-stranded DNA binding, single-stranded DNA-dependent ATPase activity |
| Conservation | Found in S. cerevisiae and conserved in eukaryotes |
What Is GO:0097505?
GO:0097505 (Rad6-Rad18 complex) is a ubiquitin ligase complex that functions in post-replicative bypass of UV-damaged DNA and UV mutagenesis. In S. cerevisiae, the complex contains the ubiquitin conjugating enzyme Rad6 and Rad18, a protein with a RING finger motif and a nucleotide binding motif. The heterodimer exhibits ubiquitin conjugating activity, binds single-stranded DNA, and has single-stranded DNA-dependent ATPase activity.
Why Is Rad6-Rad18 complex Important in Cell Biology?
The Rad6-Rad18 complex is critical for DNA damage tolerance and mutagenesis, processes that allow cells to survive replication stress but can also drive genome instability and cancer. Understanding its structure, regulation, and interactions provides insights into how cells balance survival and mutagenesis, and offers potential targets for cancer therapy.
• Mediates post-replicative bypass of UV-damaged DNA, a key DNA damage tolerance pathway.
• Monoubiquitinates PCNA to switch between error-free and error-prone lesion bypass.
• Ubiquitinates the 9-1-1 checkpoint clamp, acting as a eukaryotic SOS response.
• Interacts with translesion synthesis polymerases, such as DNA polymerase η.
• Its dysregulation is associated with cancer and chemoresistance.
• Serves as a model for studying ubiquitin signaling in DNA repair.
• Rad18 domains are functionally characterized for Rad6 binding, DNA binding, and PCNA modification.
• Oligomeric state and interactions with translesion synthesis proteins are actively studied.
Rad6-Rad18 complex: Components, Assembly and Research Methods
What Happens During Rad6-Rad18 complex?
In simple terms: The Rad6-Rad18 complex helps cells copy past DNA damage, which can cause mutations but allows survival.
The Rad6-Rad18 complex functions in post-replicative bypass of UV-damaged DNA and UV mutagenesis. It monoubiquitinates PCNA to promote DNA damage tolerance. The complex also ubiquitinates the 9-1-1 checkpoint clamp, mediating a eukaryotic SOS response. These activities are regulated by diffusion along RPA filaments.
Structure and Composition of Rad6-Rad18 complex
In simple terms: The complex is made of two proteins, Rad6 and Rad18, that stick together to form a working machine.
The Rad6-Rad18 complex is a heterodimer of Rad6, a ubiquitin-conjugating enzyme, and Rad18, which contains a RING finger motif and a nucleotide binding motif. Domains required for dimerization have been mapped. The oligomeric state of Rad6-Rad18 and its interactions with translesion synthesis proteins have been characterized.
Molecular Mechanism of Rad6-Rad18 complex
In simple terms: Rad6-Rad18 attaches ubiquitin to target proteins, using energy from ATP and binding to single-stranded DNA.
The yeast Rad6-Rad18 heterodimer has ubiquitin conjugating activity, binds single-stranded DNA, and possesses single-stranded DNA-dependent ATPase activity. Rad18 domains are functionally characterized for Rad6 binding, ubiquitin, DNA binding, and PCNA modification. PCNA monoubiquitination is regulated by diffusion of Rad6/Rad18 complexes along RPA filaments. The complex interacts with DNA polymerase η via PIP-like motifs.
Regulation of Rad6-Rad18 complex
In simple terms: The complex is controlled by how it moves along DNA and interacts with other proteins.
Rad6/Rad18 complexes diffuse along RPA filaments to regulate PCNA monoubiquitination. The complex also mediates a eukaryotic SOS response by ubiquitinating the 9-1-1 checkpoint clamp. Interactions with translesion synthesis proteins modulate its activity.
Key Genes Involved in GO:0097505 Rad6-Rad18 complex
The Rad6-Rad18 complex involves key genes and proteins that mediate DNA damage tolerance and mutagenesis.
| Gene | Major Role | Research Relevance |
|---|---|---|
| RAD6 | Ubiquitin-conjugating enzyme; forms heterodimer with Rad18 | Target for knockout to study DNA damage tolerance |
| RAD18 | RING finger protein; binds Rad6 and DNA | Point mutations in RING domain affect PCNA modification |
| PCNA | Substrate of Rad6-Rad18; monoubiquitinated | Knock-in of ubiquitin-acceptor lysine mutants |
| POL30 | Yeast PCNA; monoubiquitinated by Rad6-Rad18 | Model for PCNA modification studies |
| POLH | DNA polymerase η; interacts with Rad6-Rad18 | Knockout to study translesion synthesis |
| RAD5 | Helicase involved in error-free damage tolerance | Genetic interaction with Rad6-Rad18 |
| RAD52 | Recombination protein; error-free pathway | Epistasis analysis with Rad6-Rad18 |
| REV1 | Translesion synthesis polymerase | Interaction with Rad6-Rad18 |
| REV3 | Translesion synthesis polymerase | Error-prone pathway |
| REV7 | Subunit of Pol ζ | Error-prone pathway |
| MEC3 | 9-1-1 clamp component; ubiquitinated by Rad6-Rad18 | Checkpoint activation studies |
| DDC1 | 9-1-1 clamp component | Checkpoint activation studies |
| RAD17 | 9-1-1 clamp loader | Checkpoint activation studies |
| RPA | Single-stranded DNA binding protein; guides Rad6-Rad18 diffusion | In vitro reconstitution |
| UBI4 | Ubiquitin precursor | Ubiquitin supply for assays |
| SRS2 | Helicase; error-free damage tolerance | Genetic interaction |
| RAD55 | Recombination protein | Epistasis analysis |
How Is Rad6-Rad18 complex Regulated?
The Rad6-Rad18 complex is regulated by diffusion along RPA filaments, which controls PCNA monoubiquitination. It also mediates a eukaryotic SOS response by ubiquitinating the 9-1-1 checkpoint clamp. Interactions with translesion synthesis proteins, such as DNA polymerase η, modulate its activity.
Rad6-Rad18 complex and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| RAD6 | Cancer chemoresistance | Knockout cell lines for drug sensitivity |
| RAD18 | Cancer and genome instability | Point mutation in RING domain |
| PCNA | Cancer and DNA damage tolerance | Knock-in of ubiquitin-acceptor mutants |
| POLH | Xeroderma pigmentosum variant | Knockout for UV sensitivity |
| MEC3 | Checkpoint defects | Knockout for checkpoint activation |
Cancer and Chemoresistance
Dysregulation of Rad6-Rad18-mediated PCNA monoubiquitination is linked to cancer and chemoresistance. Targeting this complex may sensitize tumors to DNA-damaging agents.
Genome Instability
The Rad6-Rad18 complex promotes mutagenesis, which can drive genome instability and tumorigenesis.
Neurodegeneration
Defects in DNA damage tolerance pathways, including Rad6-Rad18, may contribute to neurodegeneration, though direct evidence is limited.
From Rad6-Rad18 complex-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does Rad6-Rad18 mediate UV mutagenesis? | RAD6 or RAD18 knockout yeast |
| How does Rad18 RING domain affect PCNA modification? | Point mutation in RAD18 RING domain |
| What is the role of PCNA monoubiquitination? | Knock-in of PCNA ubiquitin-acceptor mutant |
| How does Rad6-Rad18 interact with DNA polymerase η? | Tagged knock-in of POLH |
| What is the oligomeric state of Rad6-Rad18? | Overexpression and purification |
| Does Rad6-Rad18 ubiquitinate 9-1-1 clamp? | Knockout of MEC3 or DDC1 |
How to Study the Rad6-Rad18 complex Process
| Method | What It Measures | Typical Application |
|---|---|---|
| In vitro ubiquitination | Ubiquitin conjugation to PCNA | Enzymatic activity of Rad6-Rad18 |
| ATPase assay | Single-stranded DNA-dependent ATP hydrolysis | Characterization of Rad6-Rad18 |
| Electrophoretic mobility shift assay | DNA binding | Rad18 DNA binding domain |
| Fluorescence microscopy | Diffusion along RPA filaments | PCNA monoubiquitination dynamics |
| Yeast two-hybrid | Protein-protein interactions | Rad6-Rad18 dimerization |
| Mass spectrometry | Oligomeric state | Rad6-Rad18 complex composition |
| CRISPR knockout | Gene function | DNA damage sensitivity |
| CRISPR knock-in | Tagged proteins | Interaction studies |
Ubiquitination Assays
In vitro ubiquitination assays measure the transfer of ubiquitin to substrates like PCNA, using purified Rad6-Rad18 complex.
DNA Binding and ATPase Assays
Single-stranded DNA binding and ATP hydrolysis are measured to characterize Rad6-Rad18 activity.
Fluorescence Microscopy
Live-cell imaging of Rad6/Rad18 diffusion along RPA filaments reveals dynamics of PCNA monoubiquitination.
Genetic Interaction Screens
Epistasis analysis with DNA repair mutants identifies pathways involving Rad6-Rad18.
How CRISPR Can Be Used to Study GO:0097505 Rad6-Rad18 complex
Knockout
CRISPR knockout of RAD6 or RAD18 abolishes Rad6-Rad18 complex function, leading to UV sensitivity and reduced mutagenesis.
Point Mutation
Point mutations in the RING domain of RAD18 disrupt ubiquitin ligase activity and PCNA modification.
Knock-in
Knock-in of tagged Rad6 or Rad18 allows visualization and purification of the complex.
Overexpression
Overexpression of Rad6-Rad18 enables structural and biochemical studies of its oligomeric state.
How EDITGENE Supports Rad6-Rad18 complex Research
Researchers studying Rad6-Rad18 complex-related genes often need to determine whether a candidate gene is causally involved in DNA damage tolerance, mutagenesis, or cancer chemoresistance. EDITGENE provides CRISPR-based services to create precise cellular models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for Rad6-Rad18 complex research.
Frequently Asked Questions About Rad6-Rad18 complex
What is the Rad6-Rad18 complex?
The Rad6-Rad18 complex (GO:0097505) is a ubiquitin ligase complex involved in post-replicative bypass of UV-damaged DNA and UV mutagenesis.
What genes are involved in the Rad6-Rad18 complex?
The complex includes RAD6, encoding a ubiquitin-conjugating enzyme, and RAD18, encoding a RING finger and nucleotide binding protein.
What is the function of GO:0097505?
GO:0097505 functions in DNA damage tolerance by monoubiquitinating PCNA and the 9-1-1 clamp.
How is the Rad6-Rad18 complex regulated?
It is regulated by diffusion along RPA filaments and interactions with translesion synthesis proteins.
What diseases are associated with Rad6-Rad18 complex?
Dysregulation is linked to cancer and chemoresistance.
What experimental models are used to study Rad6-Rad18 complex?
Yeast knockouts, point mutants, and tagged knock-ins are common models.
What is the role of Rad18 in the complex?
Rad18 contains a RING finger and nucleotide binding motif, and is required for DNA binding and PCNA modification.
How does Rad6-Rad18 mediate UV mutagenesis?
It promotes error-prone translesion synthesis by monoubiquitinating PCNA.
What is the oligomeric state of Rad6-Rad18?
The complex forms a heterodimer and may have higher-order oligomers.
How can CRISPR help study Rad6-Rad18 complex?
CRISPR knockout, point mutation, knock-in, and overexpression models enable functional dissection.
Conclusion
The Rad6-Rad18 complex (GO:0097505) is a key ubiquitin ligase complex that mediates DNA damage tolerance and mutagenesis. Its components, Rad6 and Rad18, are conserved and functionally characterized. Studying this complex provides insights into cancer biology and genome stability, and CRISPR-based models are powerful tools for such research.
References
- 1. Li M et al.. 2020. PCNA Monoubiquitination Is Regulated by Diffusion of Rad6/Rad18 Complexes along RPA Filaments.. Biochemistry 59(49):4694-4702 PMID: 33242956
- 2. Xu X et al.. 2015. Error-free DNA-damage tolerance in Saccharomyces cerevisiae.. Mutat Res Rev Mutat Res 764:43-50 PMID: 26041265
- 3. Bailly V et al.. 1997. Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein.. Mol Cell Biol 17(8):4536-43 PMID: 9234711
- 4. Woodward TJ et al.. 2026. The Oligomeric State of Rad6-Rad18 and Its Interactions with Translesion Synthesis Proteins.. Biomolecules 16(8) PMID: 42650838
- 5. Bailly V et al.. 1997. Yeast DNA repair proteins Rad6 and Rad18 form a heterodimer that has ubiquitin conjugating, DNA binding, and ATP hydrolytic activities.. J Biol Chem 272(37):23360-5 PMID: 9287349
- 6. Fu Y et al.. 2008. Rad6-Rad18 mediates a eukaryotic SOS response by ubiquitinating the 9-1-1 checkpoint clamp.. Cell 133(4):601-11 PMID: 18485869
- 7. Ripley BM et al.. 2020. Yeast DNA polymerase η possesses two PIP-like motifs that bind PCNA and Rad6-Rad18 with different specificities.. DNA Repair (Amst) 95:102968 PMID: 32932109
- 8. Notenboom V et al.. 2007. Functional characterization of Rad18 domains for Rad6, ubiquitin, DNA binding and PCNA modification.. Nucleic Acids Res 35(17):5819-30 PMID: 17720710
