# Mapping and Visualizing Uracils Created by AID/APOBEC Enzymes using UdgX

> **NIH NIH R21** · WAYNE STATE UNIVERSITY · 2020 · $179,527

## Abstract

The creation and elimination of uracils in DNA plays a central role in the affinity maturation of B
lymphocytes. Activation-induced deaminase (AID) converts cytosines in single-stranded DNA to uracil and the
processing of these uracils by DNA repair pathways causes base substitution mutations (somatic hypermutation;
SHM) and strand breaks that lead to region-specific recombination (class-switch recombination; CSR) in
immunoglobulin (Ig) genes. AID is essential for both SHM and CSR. Most studies of SHM and CSR use
mutations, strand breaks or translocations as proxies for the uracils created by AID. This is problematic because
uracils in DNA may be repaired through error-prone or error-free pathways leading to many possible outcomes
including replacement of uracils with cytosines, transition and transversion mutations, and strand breaks. In
particular, a restoration of cytosines at these uracils erases the evidence of AID-mediated cytosine deamination.
All current technologies for detecting, quantifying and mapping uracils are indirect. We propose here
development of the first technology to directly map uracils created by the AID/APOBEC family deaminases which
contribute to both innate and acquired immunity in humans at the genomic level. Furthermore, we propose to
use it to directly visualize the uracils at a single cell level.
 A mycobacterial protein, UdgX, links covalently to uracils in DNA with a strong preference for U·G pairs.
It forms denaturation-resistant complexes with uracils and will be used to pull-down and sequence genomic DNA
fragments containing uracils. To facilitate this, the protein has been appended with FLAG and other tags, and
these tags allow for affinity pull-down of DNA-protein complexes. To demonstrate the feasibility of this approach,
we will pull down DNA fragments from uracil excision-deficient Escherichia coli expressing APOBEC3A (A3A),
prepare and sequence libraries derived from them, and map them to the E. coli genome. To determine the
accuracy of the new uracil pull-down technique, genomic map of uracils created by A3A using UdgX will be
compared with a recently created map of uracils created by A3A using an alternate pull-down technique. In a
second approach to demonstrate usefulness of this technology, UNG–/– CH12F3 cells will be stimulated to
undergo CSR and transfected with mCherry-tagged UdgX. The cells will be fixed and the Ig switch regions µ and
α will be visualized using FISH probes. A colocalization of the mCherry signal with the FISH signal will confirm
that UdgX has bound at the IgH locus. We will also use the FLAG tag to pull-down the CH12F3 genomic
fragments linked to UdgX, and use qPCR and deep sequencing to confirm that UdgX preferentially pulls down
Sµ and Sα regions. Our long-term goal is to combine the technologies created under this proposal to map uracils
created by AID during antibody maturation to V(D)J and appropriate switch regions in the Ig genes, and to off-
target sites across the whole ...

## Key facts

- **NIH application ID:** 9872122
- **Project number:** 5R21AI144708-02
- **Recipient organization:** WAYNE STATE UNIVERSITY
- **Principal Investigator:** ASHOK S BHAGWAT
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $179,527
- **Award type:** 5
- **Project period:** 2019-02-13 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9872122

## Citation

> US National Institutes of Health, RePORTER application 9872122, Mapping and Visualizing Uracils Created by AID/APOBEC Enzymes using UdgX (5R21AI144708-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9872122. Licensed CC0.

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