# DNA Adduct-Induced Mutagenesis

> **NIH NIH R56** · VANDERBILT UNIVERSITY · 2024 · $250,000

## Abstract

Abstract: Environmental exposures to aflatoxin B1 (AFB1), a mycotoxin isolated from Aspergillus
flavus, are linked to the etiology of hepatocellular cancer (HCC). We propose three aims. The
first examines the role of base excision repair (BER) by the hNEIL1 glycosylase, and "cancer-
prone" variants of hNEIL1 in regions of exposure to AFB1, in excising AFB1 damage from DNA.
Deficiencies in repair of AFB1 DNA damage may be associated with early onset HCC, common
in areas of China with high AFB1 exposures, and contribute to the mutagenic signature
associated with AFB1 exposures. We will employ genetically edited mouse embryonic fibroblasts
(MEFs) to determine how hNEIL1 variants modulate the mutagenic signature. We will employ
crystallography to understand mechanism for repair of AFB1 damage in DNA by hNEIL1 and
variants. Structural and dynamic changes associated with variants may reveal genetic risk
factors for HCC. Our second aim focuses on the mechanism of error-prone DNA replication of
AFB1 damage. The polymerase ζ specializes in extension beyond DNA damage and factors in
the genesis of A to T transversions characteristic of AFB1 exposures. We will employ cryo-EM to
determine structures of pol ζ complexed with AFB1-damaged DNA, accompanied by functional
assays. Crystallography will be employed to probe the mechanism of nucleotide insertion
opposite AFB1 lesions by DNA polymerase pol η. Our third aim focuses on the respective roles
of sequence-specific lesion bypass and formation of AFB1-Fapy-dG lesions in the etiology of
“Signature 24”, a sequence-specific pattern of G to T transversions characteristic of exposures
to AFB1. Fidelity and efficiency of bypass will be tested using sequence-specific substrates
including modeling replication forks. We will examine how the AFB1 mutagenic signature is
altered in Pol ζ-deficient MEFs. Sequence-specific rates of adduct formation will be probed by
competition assays under conditions in which AFB1-epoxide is the limiting reagent. We will
determine if re-arrangement of AFB1-N7-dG to (a) less mutagenic AP sites, or (b) mutagenic
AFB1-Fapy-dG, depends upon DNA sequence. We will examine cytosine methylation to probe
epigenetic contributions to the AFB1 mutagenic signature. We will employ NMR to monitor
complex sequence-specific conformational and configurational equilibria associated with AFB1-
Fapy-dG lesions in dsDNAs, in situ. We will also use NMR to measure sequence-specific
kinetics of DNA base pair opening, which may control rates of base-flipping during repair. AFB1
is an important environmental genotoxin; our work should provide new understanding of
environmental determinants of HCC.

## Key facts

- **NIH application ID:** 11089140
- **Project number:** 2R56ES029357-30A1
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** Michael P Stone
- **Activity code:** R56 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $250,000
- **Award type:** 2
- **Project period:** 1992-02-15 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11089140

## Citation

> US National Institutes of Health, RePORTER application 11089140, DNA Adduct-Induced Mutagenesis (2R56ES029357-30A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/11089140. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*