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(http://www.niehs.nih.gov//portfolio/index.cfm?do=portfolio.grantdetail&&grant_number=R56ES029357&format=word)
| Principal Investigator: Stone, Michael P | |
|---|---|
| Institute Receiving Award | Vanderbilt University |
| Location | Nashville, TN |
| Grant Number | R56ES029357 |
| Funding Organization | National Institute of Environmental Health Sciences |
| Award Funding Period | 15 Feb 1992 to 31 May 2025 |
| DESCRIPTION (provided by applicant): | 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. |
| Science Code(s)/Area of Science(s) |
Primary: 09 - Genome Integrity Secondary: - |
| Publications | No publications associated with this grant |
| Program Officer | Daniel Shaughnessy |