REV1 and DHX36 Coordinate G-Quadruplex DNA Replication and Tolerance

Study Background and Research Question

G-quadruplex (G4) DNA structures, formed by guanine-rich sequences, present a formidable barrier to DNA replication due to their stable, non-canonical four-stranded conformations. These structures are implicated in genome instability and are known to challenge the progression of replication forks, posing risks for mutagenesis and incomplete DNA synthesis. Despite evidence that translesion synthesis (TLS) polymerases can bypass such obstacles, the precise orchestration between the DNA replication machinery, helicases, and TLS polymerases at G4 sites has remained unclear. The reference study by Ketkar et al. (Nucleic Acids Research, 2026) directly addresses how human REV1, a Y-family TLS polymerase, interacts with the G4-resolving helicase DHX36 to manage replication and damage tolerance at G4 DNA motifs.

Key Innovation from the Reference Study

The central innovation of this research is the delineation of a direct, functionally critical interaction between the C-terminal domain of REV1 and a newly defined C-terminal region of DHX36. This interaction acts as a two-tiered regulatory mechanism: first, by coordinating helicase-dependent G4 DNA unwinding, and second, by suppressing the formation of single-stranded DNA (ssDNA) gaps during G4 stabilization. The study not only characterizes the molecular interface between REV1 and DHX36 but also shows its pivotal role in maintaining replication fork integrity and genome stability in the face of stabilized G4 DNA structures.

Methods and Experimental Design Insights

To dissect the REV1-DHX36 axis, the research combines genetic, biochemical, and cell biological approaches:

• CRISPR/Cas9-mediated knockout and rescue experiments in human cell lines to assess the impact of REV1 and DHX36 perturbation on replication dynamics at G4 sites.
• Use of pyridostatin (PDS), a small molecule G4 stabilizer, to challenge fork progression and induce G4-dependent replication stress.
• Single-molecule fiber assays and immunofluorescence to visualize replication fork progression and nuclear G4 accumulation.
• Co-immunoprecipitation and in vitro binding assays to map the physical interface between REV1 and DHX36.
• Assessment of mutation frequency and strand-specific mutagenesis to dissect the functional consequences of REV1 loss during G4 DNA replication.

These complementary approaches allowed the authors to interrogate both the structural and functional aspects of the REV1-DHX36 partnership in G4 DNA tolerance and repair.

Core Findings and Why They Matter

The study's findings clarify several mechanistic aspects of G4 DNA replication and repair:

• REV1 is essential for replication fork progression at G4 DNA. Loss of REV1 leads to a switch from canonical replication to a PrimPol-dependent mechanism, which is less effective at suppressing ssDNA gap formation—especially under G4-stabilizing conditions (Ketkar et al., 2026).
• Strand-specific effects: Mutagenic replication of G4 DNA is more impaired on the leading strand in the absence of REV1, while lagging strand mutagenesis is particularly sensitive to G4 stabilization by agents like PDS.
• REV1 deficiency amplifies DNA damage signaling. Cells lacking REV1 show increased nuclear G4 signals and enhanced activation of the ATM/ATR DNA damage response pathways.
• REV1 interacts directly with DHX36. The interaction requires a specific C-terminal region of DHX36, and disruption of this interface leads to uncoupling of DHX36 from REV1 and the replication machinery during prolonged G4 stabilization. This uncoupling correlates with the accumulation of helicase at sites distal to replication forks and increased genomic instability.
• Two-tiered mechanism: REV1 coordinates helicase-dependent G4 unwinding and acts to prevent ssDNA gap accumulation, thus safeguarding fork integrity during replication stress.

These insights underscore the importance of protein-protein interactions in managing replication challenges posed by non-canonical DNA structures. The findings have broader implications for understanding genome maintenance, the cellular response to DNA damage, and the etiology of diseases linked to G4 instability, such as cancer.

Comparison with Existing Internal Articles

The current study complements and extends knowledge outlined in the internal resource, "AZD1390: Practical Guide to ATM Kinase Inhibitor Use in DNA Repair Studies", which focuses on the use of selective ATM kinase inhibitors like AZD1390 to dissect DNA double-strand break (DSB) repair and radiosensitization pathways. While AZD1390 research is centered on ATM-mediated signaling and cellular responses to DSBs, the reference paper by Ketkar et al. highlights upstream events at the replication fork—specifically, the interplay between DNA polymerases, helicases, and replication obstacles like G4 DNA. Notably, both lines of evidence converge on the importance of coordination among DNA repair proteins and signaling pathways, as REV1 deficiency leads to amplified ATM/ATR signaling, suggesting that tools like AZD1390 can further probe these downstream responses in the context of G4-induced genome stress.

Limitations and Transferability

Several caveats should be considered when interpreting the results of this study:

• Cell line specificity: Most experiments were performed in human cancer cell lines; the transferability to primary or non-transformed cells requires further validation.
• Acute versus chronic G4 stabilization: The use of PDS and other G4 stabilizers models acute perturbation, which may not fully recapitulate endogenous G4 challenges during unperturbed replication.
• Functional redundancy: Other helicases and TLS polymerases may compensate for the loss of REV1 or DHX36 in certain contexts, potentially masking broader phenotypes.
• Genomic context: Effects may vary depending on the genomic location and density of G4 motifs, as well as the transcriptional environment.

Despite these limitations, the mechanistic insights provided by the defined REV1-DHX36 interface and its role in G4 DNA tolerance are likely to be broadly relevant to fields investigating DNA replication stress, genome instability, and therapeutic targeting of DNA repair pathways.

Research Support Resources

For researchers aiming to probe ATM signaling and its role in the DNA damage response—particularly in the context of replication stress and G4-induced DNA damage—tools such as AZD1390 (SKU B8328) offer a highly selective means of inhibiting ATM kinase activity. According to the product information, AZD1390 is effective in cellular models at nanomolar concentrations, radiosensitizes glioma and lung cancer cell lines, and is suited for studies focused on DNA double-strand break repair and checkpoint signaling. When designing experiments to dissect the interplay between DNA replication obstacles, repair proteins, and damage response signaling, incorporating a potent ATM kinase inhibitor can help clarify the downstream effects of genome stress uncovered by studies like that of Ketkar et al.

Protocol Parameters

• AZD1390 dosing for ATM inhibition: Use 3 nM in glioblastoma LN18 cells for robust ATM inhibition; for NCI-H2228 lung cancer cells, 10 nM AZD1390 in combination with radiation effectively induces G2 arrest and apoptosis (see product details).
• Solubility considerations: Dissolve AZD1390 in DMSO (≥19.6 mg/mL) or ethanol (≥3.04 mg/mL) with gentle warming and ultrasonic treatment; avoid long-term storage of prepared solutions.
• Model selection: For studies on G4 DNA or replication stress, select cell lines with characterized G4 motif content and/or known repair pathway deficiencies.

As with all DNA repair studies, careful experimental design—including choice of cell model, dosing regimen, and endpoint assays—is essential for reproducible and interpretable results. APExBIO supplies AZD1390 in high purity for research use, with storage and preparation guidance provided online.