GO:0017125 deoxycytidyl transferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0017125 deoxycytidyl transferase activity is a molecular function defined as the catalysis of dCMP insertion opposite a template abasic site in DNA.
• The founding enzyme for this activity is yeast Rev1p, which was shown to possess deoxycytidyl transferase activity in 1996.
• Human REV1 carries the same activity, and it is closely associated with the conserved polymerase domain of the protein.
• This activity is a key step in translesion synthesis, allowing replication to bypass abasic sites and other lesions.
• REV1 and its deoxycytidyl transferase activity cooperate with DNA polymerase zeta and influence mutagenesis in eukaryotes.
• Dysregulation of REV1-mediated bypass is linked to alkylation damage tolerance, chromosome stability, and cancer biology.
Description
Deoxycytidyl transferase activity (GO:0017125) is a specialized DNA polymerase activity that inserts a deoxycytidine monophosphate (dCMP) residue opposite a template abasic site in DNA. This activity is best known from the REV1 protein, a Y-family translesion synthesis polymerase that was first demonstrated to catalyze this reaction in yeast. Unlike replicative polymerases, which stall at non-coding lesions, REV1 can use the abasic site as a template and incorporate dCMP, thereby allowing DNA synthesis to continue past the damage. The reaction is template-dependent and requires a free 3'-hydroxyl group on the primer strand, but it does not require the template base to be a canonical nucleotide. Because abasic sites are among the most common spontaneous and induced DNA lesions, this activity has broad implications for genome stability, mutagenesis, and cellular survival after DNA damage. Researchers study GO:0017125 to understand how cells tolerate unrepaired DNA lesions, how mutations arise during translesion synthesis, and how REV1-dependent bypass can be targeted in cancer therapy.
deoxycytidyl transferase activity At A Glance
| GO ID | GO:0017125 |
|---|---|
| GO term | deoxycytidyl transferase activity |
| Ontology | molecular_function |
| Synonym | deoxycytidyl transferase activity, template-dependent |
| Definition | Catalysis of the insertion of a dCMP residue opposite a template abasic site in DNA. |
| Major function | Translesion synthesis past abasic sites by incorporating dCMP opposite the lesion. |
| Representative enzyme | REV1 (yeast Rev1p and human REV1) |
| Pathway context | DNA damage tolerance and mutagenesis |
| Cofactor requirement | Divalent metal ions typical of DNA polymerases (inferred from polymerase domain) |
What Is GO:0017125?
GO:0017125 deoxycytidyl transferase activity is defined by QuickGO as the catalysis of the insertion of a dCMP residue opposite a template abasic site in DNA. In other words, the enzyme reads an abasic site, which lacks a coding base, and preferentially adds a cytosine nucleotide to the growing DNA strand opposite that site. This is a template-dependent reaction because the enzyme still uses the damaged DNA strand as a guide, even though the template position is non-informational. The activity is a molecular function and is distinct from canonical DNA polymerase activity because it is specialized for bypassing abasic sites rather than replicating normal templates.
Why Is deoxycytidyl transferase activity Important in Cell Biology?
GO:0017125 is important because it defines a non-canonical DNA synthesis reaction that allows cells to survive replication-blocking lesions. Abasic sites are frequent DNA lesions that can arise spontaneously or from chemical exposure, and if they are not bypassed, replication forks can collapse, leading to chromosome breakage and cell death. The deoxycytidyl transferase activity of REV1 provides a mechanism to insert a nucleotide opposite an abasic site, enabling completion of DNA replication at the cost of potential mutations. This activity is therefore central to the balance between genome stability and mutagenesis, and it is a potential target for modulating chemotherapy and understanding cancer predisposition.
• Provides a mechanism for bypassing abasic sites, which are among the most common DNA lesions.
• Contributes to mutagenesis because inserting dCMP opposite a non-informational lesion can fix mutations.
• Supports cell survival after DNA-damaging chemotherapy and radiation.
• Cooperates with DNA polymerase zeta to extend from the inserted nucleotide.
• Is conserved from yeast to humans, making model organisms informative.
• Plays a role in tolerance to alkylation damage and prevention of chromosome shattering.
• Is a candidate target for cancer therapy because REV1-dependent bypass can promote resistance.
• Helps explain how cells tolerate unrepaired DNA damage during S phase.
• Links DNA repair, replication, and mutagenesis pathways in eukaryotes.
• Provides a biochemical assay for measuring translesion synthesis activity.
Molecular Mechanism of deoxycytidyl transferase activity
Substrate recognition and abasic site binding
In simple terms: The enzyme first finds the damaged spot in DNA where a base is missing.
REV1 binds to DNA containing an abasic site and positions the lesion in its active site. The enzyme recognizes the non-informational template position and prepares to insert a nucleotide opposite it. This binding step is template-dependent, meaning the enzyme still uses the damaged strand as a guide even though the template base is absent.
Catalytic insertion of dCMP
In simple terms: The enzyme adds a cytosine nucleotide across from the missing base.
The deoxycytidyl transferase activity catalyzes the transfer of dCMP from a dCTP substrate to the 3'-hydroxyl end of the primer strand, opposite the abasic site. This reaction is the defining catalytic step of GO:0017125 and is closely associated with the conserved polymerase domain of REV1. The insertion is preferential for dCMP, which is why the activity is named deoxycytidyl transferase.
Role of the conserved polymerase domain
In simple terms: A specific part of the REV1 protein carries out the chemistry.
The human REV1 protein contains a conserved polymerase domain that is required for its deoxycytidyl transferase activity. Mutations in this domain can abolish the ability to insert dCMP opposite an abasic site, demonstrating that the catalytic core is responsible for the reaction. This domain is also important for the protein's function in translesion synthesis in cells.
Coordination with DNA polymerase zeta
In simple terms: After REV1 adds the first nucleotide, another polymerase extends the DNA chain.
REV1-mediated insertion of dCMP opposite an abasic site is often followed by extension of the primer by DNA polymerase zeta. This cooperation is required for efficient bypass of the lesion and for mutagenesis in eukaryotes. Recent work shows that Rev1 can both promote and regulate polymerase zeta activity on damaged versus undamaged DNA, indicating a complex regulatory interplay.
Template dependence and lesion bypass
In simple terms: The enzyme still reads the damaged DNA strand, but the missing base means it inserts a default nucleotide.
The reaction is template-dependent because the enzyme uses the abasic site-containing strand as the template, but the absence of a coding base leads to preferential insertion of dCMP. This bypass mechanism allows replication to continue past the lesion, but it can also introduce mutations if the original base was not cytosine. The activity is therefore a double-edged sword: it promotes survival but can increase mutagenesis.
Key Genes Involved in GO:0017125 deoxycytidyl transferase activity
The following genes and proteins are directly implicated in deoxycytidyl transferase activity (GO:0017125) or in the translesion synthesis pathway that depends on it.
| Gene | Major Role | Research Relevance |
|---|---|---|
| REV1 | Primary enzyme with deoxycytidyl transferase activity; inserts dCMP opposite abasic sites | Core gene for GO:0017125; knockout and point mutations used to study bypass and mutagenesis |
| REV3L | Catalytic subunit of DNA polymerase zeta; extends from REV1-inserted nucleotides | Cooperates with REV1 in translesion synthesis; knockout models show reduced mutagenesis |
| REV7 | Accessory subunit of DNA polymerase zeta; interacts with REV1 | Required for polymerase zeta function; studied in knockout and knock-in models |
| POLH | Y-family polymerase that bypasses UV lesions | Related translesion polymerase; comparative studies with REV1 |
| POLI | Y-family polymerase involved in lesion bypass | May cooperate with REV1 in some contexts; knockout models available |
| POLK | Y-family polymerase that bypasses bulky lesions | Related bypass polymerase; used in comparative studies |
| PCNA | Sliding clamp that coordinates translesion synthesis | Regulates recruitment of REV1 and other polymerases to stalled forks |
| RAD18 | E3 ubiquitin ligase that monoubiquitinates PCNA | Upstream regulator of translesion synthesis; knockout affects REV1 recruitment |
| UBE2B | E2 ubiquitin-conjugating enzyme for PCNA monoubiquitination | Works with RAD18; knockout models show defective bypass |
| ATR | DNA damage response kinase | Regulates cell cycle and translesion synthesis after damage |
| ATRIP | ATR-interacting protein | Part of ATR checkpoint; affects REV1-dependent tolerance |
| TP53 | Tumor suppressor that coordinates DNA damage responses | Loss alters reliance on REV1-mediated bypass; relevant to cancer models |
| BRCA1 | Homologous recombination factor | Loss may increase dependence on REV1; synthetic lethality studies |
| BRCA2 | Homologous recombination factor | Similar to BRCA1; potential combination with REV1 inhibition |
| FANCD2 | Fanconi anemia protein involved in crosslink repair | May intersect with REV1 pathway; knockout models |
| MCM2 | Replicative helicase component | Affects fork progression and need for bypass |
| POLA1 | Replicative polymerase alpha | Stalls at abasic sites; REV1 provides bypass |
| POLB | Base excision repair polymerase | Processes abasic sites; competition with REV1 |
How Is deoxycytidyl transferase activity Regulated?
The deoxycytidyl transferase activity of REV1 is regulated at multiple levels. Its recruitment to stalled replication forks depends on PCNA monoubiquitination by RAD18 and UBE2B, which creates a binding site for REV1. The activity is also influenced by the DNA damage response kinase ATR, which coordinates cell cycle arrest and translesion synthesis. Recent evidence indicates that Rev1 can both promote and restrain DNA polymerase zeta activity on damaged versus undamaged DNA, suggesting a dynamic regulatory role. In addition, the catalytic activity of human REV1 on undamaged and damaged DNA is modulated by the template context and lesion type. These regulatory layers ensure that deoxycytidyl transferase activity is deployed primarily when replication forks encounter lesions, minimizing mutagenesis on normal templates.
deoxycytidyl transferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| REV1 | Chemotherapy resistance; alkylation damage tolerance | REV1 knockout and point-mutation cell lines treated with alkylating agents |
| REV3L | Mutagenesis and cancer progression | REV3L knockout models to assess translesion synthesis |
| BRCA1 | Homologous recombination deficiency; synthetic lethality with REV1 | BRCA1-mutant cells with REV1 knockdown |
| BRCA2 | Homologous recombination deficiency; potential REV1 dependency | BRCA2-mutant cells with REV1 inhibition |
| TP53 | Loss of checkpoint control; increased mutagenesis | TP53-null cells with REV1 overexpression or knockout |
Cancer and chemotherapy resistance
REV1-mediated deoxycytidyl transferase activity contributes to tolerance of DNA-damaging chemotherapy by allowing replication past abasic sites and other lesions. Cancer cells with high REV1 activity may survive alkylating agents and platinum-based drugs more effectively, making REV1 a candidate therapeutic target. Loss of REV1 can sensitize cells to these agents, and combining REV1 inhibition with existing chemotherapy is an active area of research.
Genome instability and chromosome shattering
In Drosophila melanogaster, REV1 coordinates a multi-faceted tolerance response to DNA alkylation damage and prevents chromosome shattering. This suggests that loss of deoxycytidyl transferase activity can lead to catastrophic genome instability when cells are exposed to alkylating agents. The findings highlight the importance of REV1-dependent bypass for maintaining chromosome integrity under genotoxic stress.
Mutagenesis and cancer predisposition
Because deoxycytidyl transferase activity inserts dCMP opposite a non-informational lesion, it can introduce mutations that contribute to cancer initiation. In eukaryotes, mutagenesis dependent on DNA polymerase zeta and Rev1p is well documented. Dysregulation of this pathway may increase mutation rates and promote tumor heterogeneity, making it relevant to cancer predisposition and progression.
From deoxycytidyl transferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of REV1 deoxycytidyl transferase activity sensitize cells to alkylating agents? | REV1 knockout cell line |
| Which catalytic residues are required for dCMP insertion? | Point-mutation knock-in of REV1 catalytic domain |
| Does a disease-associated REV1 variant alter bypass activity? | Knock-in of the variant into the endogenous locus |
| Where and when is REV1 recruited to damage sites? | Tagged knock-in of REV1 with fluorescent protein |
| Does REV1 overexpression increase mutagenesis? | Overexpression cell model with mutation reporter |
| Can REV1 inhibition synergize with chemotherapy? | REV1 knockout or point-mutation cells treated with drugs |
How to Study the deoxycytidyl transferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Primer extension assay | Insertion of dCMP opposite an abasic site | Biochemical characterization of REV1 activity |
| Gel electrophoresis | Size and amount of extended primer | Quantifying bypass efficiency |
| Site-specific lesion plasmid | Cellular bypass and mutagenesis | Translesion synthesis reporter assays |
| CRISPR knockout | Loss of REV1 function | Sensitization to DNA-damaging agents |
| Point-mutation knock-in | Catalytic residue requirement | Structure-function studies |
| Fluorescent tagging | Subcellular localization and recruitment | Live-cell imaging of REV1 |
| RNA-seq | Transcriptional changes after damage | Pathway analysis in REV1 mutants |
| CRISPR library screen | Genome-wide modifiers of drug sensitivity | Identifying synthetic lethal interactions |
Biochemical assays for deoxycytidyl transferase activity
The activity can be measured in vitro using primer extension assays with defined DNA templates containing a site-specific abasic site. Incorporation of radiolabeled or fluorescently labeled dCMP opposite the lesion is quantified by gel electrophoresis. Such assays were used to demonstrate the activity of yeast and human REV1 and to map it to the conserved polymerase domain.
Cell-based translesion synthesis reporters
Plasmid- or genome-based reporters containing a single abasic site can be used to measure bypass efficiency and mutagenesis in cells. Knockout or point-mutation of REV1 reduces bypass and alters mutation spectra. These reporters allow researchers to link biochemical activity to cellular outcomes.
Genome-wide screening and bioinformatics
CRISPR library screening can identify genes that modify sensitivity to DNA-damaging agents in REV1-proficient versus REV1-deficient cells. Bioinformatics analysis of mutation signatures and gene expression can reveal pathways that depend on deoxycytidyl transferase activity. Such approaches help place GO:0017125 in the broader DNA damage response network.
Structural and computational studies
Structural modeling and molecular dynamics simulations based on the REV1 polymerase domain can predict how the enzyme accommodates an abasic site and selects dCMP. These methods complement biochemical data and guide mutagenesis experiments. Computational analysis of REV1 variants can also prioritize residues for functional testing.
How CRISPR Can Be Used to Study GO:0017125 deoxycytidyl transferase activity
Knockout
CRISPR knockout of REV1 eliminates deoxycytidyl transferase activity and allows researchers to test its role in DNA damage tolerance. REV1 knockout cells show increased sensitivity to alkylating agents and reduced translesion synthesis. Knockout models are also used to assess chromosome stability and mutagenesis.
Point Mutation
Point mutations in the conserved polymerase domain of REV1 can abolish catalytic activity while preserving protein interactions. Such knock-in models are valuable for separating the catalytic function from scaffolding roles. They help determine which phenotypes depend specifically on GO:0017125.
Knock-in
Knock-in of tagged or disease-associated REV1 variants allows precise measurement of expression, localization, and activity. Fluorescent knock-in models enable live-cell imaging of REV1 recruitment to damage sites. Knock-in of patient-derived variants can reveal functional consequences.
Overexpression
Overexpression of REV1 can increase deoxycytidyl transferase activity and mutagenesis in reporter assays. Such models are used to test whether elevated bypass activity promotes resistance to chemotherapy. Overexpression combined with DNA-damaging agents helps define the threshold for toxicity.
How EDITGENE Supports deoxycytidyl transferase activity Research
Researchers studying deoxycytidyl transferase activity-related genes often need to determine whether a candidate gene is causally involved in lesion bypass, mutagenesis, or drug resistance. EDITGENE provides CRISPR-based cell model services that enable precise manipulation of REV1 and related genes to answer these questions.
Contact EDITGENE today to design your custom CRISPR model for deoxycytidyl transferase activity research.
Frequently Asked Questions About deoxycytidyl transferase activity
What is deoxycytidyl transferase activity?
It is a molecular function defined as the catalysis of dCMP insertion opposite a template abasic site in DNA, best known from the REV1 protein.
What is GO:0017125?
GO:0017125 is the Gene Ontology identifier for deoxycytidyl transferase activity, a molecular function in DNA translesion synthesis.
What genes are involved in deoxycytidyl transferase activity?
The primary gene is REV1, which encodes the enzyme; REV3L and REV7 cooperate in the downstream extension step.
Which enzyme has deoxycytidyl transferase activity?
Yeast Rev1p and human REV1 are the founding enzymes with this activity.
How is deoxycytidyl transferase activity measured?
It is measured by primer extension assays using DNA templates with a site-specific abasic site and labeled dCTP.
Why is deoxycytidyl transferase activity important for cancer?
It allows cancer cells to bypass DNA damage and survive chemotherapy, making REV1 a potential drug target.
What diseases are linked to REV1 and deoxycytidyl transferase activity?
Cancer chemotherapy resistance and genome instability disorders are linked to REV1 function.
Does deoxycytidyl transferase activity require a template?
Yes, it is template-dependent because the enzyme uses the damaged DNA strand as a guide, even though the template base is absent.
What is the difference between REV1 and DNA polymerase zeta?
REV1 inserts dCMP opposite the lesion, while DNA polymerase zeta extends the primer after insertion.
How can CRISPR help study deoxycytidyl transferase activity?
CRISPR knockout, point mutation, and knock-in models allow precise testing of REV1 function in cells.
Conclusion
GO:0017125 deoxycytidyl transferase activity defines a specialized DNA synthesis reaction that inserts dCMP opposite abasic sites, enabling cells to tolerate otherwise blocking lesions. This activity, carried out by REV1, is conserved from yeast to humans and is tightly coordinated with DNA polymerase zeta and the DNA damage response. Its dual role in survival and mutagenesis makes it a compelling target for cancer research and a key node in genome stability pathways. Continued studies using CRISPR models and biochemical assays will clarify how this activity can be modulated for therapeutic benefit.
References
- 1. Nelson JR et al.. 1996. Deoxycytidyl transferase activity of yeast REV1 protein.. Nature 382(6593):729-31 PMID: 8751446
- 2. Masuda Y et al.. 2001. Deoxycytidyl transferase activity of the human REV1 protein is closely associated with the conserved polymerase domain.. J Biol Chem 276(18):15051-8 PMID: 11278384
- 3. Otsuka C et al.. 2002. The role of deoxycytidyl transferase activity of yeast Rev1 protein in the bypass of abasic sites.. Nucleic Acids Res Suppl PMID: 12903118
- 4. Bezalel-Buch R et al.. 2026. Opposing regulation by Rev1 of DNA polymerase zeta activity on damaged versus undamaged DNA.. Nucleic Acids Res 54(11) PMID: 42268683
- 5. Lawrence CW et al.. 2001. Mutagenesis in eukaryotes dependent on DNA polymerase zeta and Rev1p.. Philos Trans R Soc Lond B Biol Sci 356(1405):41-6 PMID: 11205328
- 6. Stolyarenko AD et al.. 2024. The Catalytic Activity of Human REV1 on Undamaged and Damaged DNA.. Int J Mol Sci 25(7) PMID: 38612916
- 8. Khodaverdian V et al.. 2024. REV1 Coordinates a Multi-Faceted Tolerance Response to DNA Alkylation Damage and Prevents Chromosome Shattering in Drosophila melanogaster.. bioRxiv PMID: 38405884
