# Molecular mechanisms of bacterial immune signaling through DNA damage

> **NIH NIH F31** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2024 · $25,103

## Abstract

PROJECT SUMMARY
Molecular mechanisms of bacterial immune signaling through DNA damage
 The availability of tens of thousands of bacterial genome sequences, plus new bioinformatics tools and
new understanding of bacterial genome organization, has enabled the discovery and experimental
characterization of dozens of anti-bacteriophage and anti-plasmid defense systems in bacteria. Since a typical
bacterial genome encodes 3-6 distinct defense systems, a key question is whether and how these systems can
coordinate their activities to synergistically fight an infection. In prior work on the widespread and diverse
CBASS (Cyclic oligonucleotide-Based Anti-phage Signaling System) defense systems, we identified two
transcriptional regulators – CapW and the two-protein CapH+CapP system – that boost CBASS gene
expression in response to DNA damage. Together, CapW and CapH+CapP are associated with ~10% of
CBASS systems, and are also found adjacent to a broad range of known and predicted bacterial defense
systems including Pycsar, DISARM, and BREX. These findings suggest that CapW and CapH+CapP may
mediate activation of antiviral defense in response to a universal signal of cell stress, DNA damage. Here, I will
first identify the small-molecule or nucleic acid ligand that binds and activates CapW upon DNA damage. I will
combine biochemical assays for CapW binding to both its target DNA and its ligand with x-ray crystallography
to characterize the conformational changes imposed by the ligand to control CapW-DNA binding. This work will
establish a mechanism for CapW, a widespread bacterial transcription factor. Next, I will test the idea that
CapW and CapH+CapP mediate cooperation between antiviral defense systems by sensing DNA damage.
Specifically, we hypothesize that DNA-targeting immune systems like restriction-modification and CRISPR-Cas
create DNA damage that is sensed by CapW or CapH+CapP to activate a secondary defense system (CBASS
or others) to reinforce the defensive response. I will systematically test this model by infecting cells encoding
both a restriction-modification system and a CapW- or CapH+CapP-associated CBASS system to determine if
the combination of these systems yields synergistic antiviral immunity. Additionally, I will test whether DNA
damage sensing plays a role in defense-system synergy, using structure-based mutations to either CapW or
CapP that eliminate DNA damage sensing. Together, these experiments will reveal the molecular mechanism
of CapW, and the role of DNA damage sensors in mediating synergy in bacterial defense systems. The
findings have the potential to establish a new paradigm in which DNA targeting defense systems constitute a
first line of antiviral defense, and DNA damage-activated systems constitute a second line of defense with
orthogonal mechanisms. Thus, instead of viewing bacterial defense systems in isolation, this work will
establish how they cooperate to compose a comprehensive bacterial “immune system”.

## Key facts

- **NIH application ID:** 10928707
- **Project number:** 5F31GM150210-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** Chelsea Lee Blankenchip
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $25,103
- **Award type:** 5
- **Project period:** 2023-07-01 → 2024-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10928707

## Citation

> US National Institutes of Health, RePORTER application 10928707, Molecular mechanisms of bacterial immune signaling through DNA damage (5F31GM150210-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10928707. Licensed CC0.

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