# Deoxyuridine Contamination and Innate Immune Signaling

> **NIH CA R01** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2026 · $644,234

## Abstract

ABSTRACT
Most chemotherapies target DNA replication, and their efficacy depends on the DNA damage response that
integrates DNA repair with the cell cycle. Widely used standard-of-care antimetabolites inhibit thymidylate
synthase and increase the incorporation of deoxyuridine (dU) into DNA by polymerases. dU contamination in
DNA is limited by the DDR kinase ATR that induces cell cycle checkpoints and reduces the rate of DNA
replication. This project investigates the impact of dU contamination on innate immune responses. ATR
inhibitors (ATRi) induce origin firing across active replicons and cause ribonuclease reductase degradation in
otherwise unperturbed cells. This increases both the amount of DNA replication and the amount of free dUTP
in cells. Thus, ATRi increase the incorporation of dU into DNA by polymerases. Since ATRi also inhibit cell
cycle checkpoints, ATRi induce more dU contamination than antimetabolites. Our preliminary data show that
ATRi-induced IFN-β is dependent on uracil DNA glycosylase (UNG) which removes dU from DNA. Our data
are consistent with a model in which UNG-dependent base excision repair (BER) removes dU from DNA and
generates cytoplasmic dsDNA that induces IFN-α/β. Unexpectedly, however, we did not observe a difference in
the amount of cytoplasmic dsDNA in wild-type (WT) and UNG knockout (∆UNG) cells after treatment with ATRi.
We reasoned that cytoplasmic DNA in ∆UNG cells may not be recognized by pattern recognition receptors
because it is dA:dU-rich. Indeed, replacing just 20 thymidine bases (T) with dU in a 200 bp dsDNA fragment
significantly decreased IFN-β induction in cells. Our data suggest that the integrity of dA:T base pairs is critical
for the IFN-α/β response in cells treated with chemotherapy and ionizing radiation (XRT). cGAS recognizes
DNA in a sequence-independent manner. However, dsDNA fragments are transcribed in the cytoplasm by RNA
Polymerase III (Pol III) to generate RNA that is recognized by RIG1. It was recently sh

## Key facts

- **NIH application ID:** 11311924
- **Project number:** 5R01CA294651-02
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** CHRISTOPHER J. BAKKENIST
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** CA
- **Fiscal year:** 2026
- **Award amount:** $644,234
- **Award type:** 5
- **Project period:** 2025-04-03T00:00:00 → 2030-03-31T00:00:00

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11311924

## Citation

> US National Institutes of Health, RePORTER application 11311924, Deoxyuridine Contamination and Innate Immune Signaling (5R01CA294651-02). Retrieved via AI Analytics 2026-07-13 from https://api.ai-analytics.org/grant/nih/11311924. Licensed CC0.

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