This project defines the responses of cells to DNA and RNA damaging agents. The work relates the structures of specific types of nucleic acid damage with the mutations those lesions produce in vivo. Two key features of the project are (1) its emphasis on in vivo analysis of the biology of DNA adducts, many of which are produced by human carcinogens and (2) the emphasis on rigorous synthetic methodology for preparation of adduct containing oligonucleotides and genomes. The work on the path ahead will divide into the following areas, which are a continuation ofthe theme established during the first four years of support. Our first goal, will be to continue to develop methodology for synthesis of DNA oligonucleotides containing specific modified bases at defined sites. The work proposed requires synthesis of oligonucleotides containing 5-substituted cytosines (produced by oxidative stress), a series of N and 0-alkylated purines and pyrimidines (produced by alkylation and inflammation), and N2 and C8-purine aromatic amine adducts (produed by tobacco smoke). In addition, we are preparing oligonucleotides containing a {guanine Nl-ethyl- N3 cytosine} interstrand crosslink and the DNA adducts of the liver carcinogen, aflatoxin 81. Our second goal is to continue the Biological evaluation of damaged DNA bases in vivo. Genomes of viruses and plasmids will be re-engineered to contain gaps (e.g., 16 nucleotides) at specific sites. The modified oligonucleotides will be insert:ed by ligation into the gaps, forming a site-specifically modified genome. To study mutation and lethality, the genomes containing defined lesions will be introduced into cells, and progeny will be isolated, enumerated to determine toxicity, and sequenced to determine mutation. Our third goal is to establish a system that permits the study of RNA lesion replication and repair in vivo. It is logical to assume that cells have a repair system to address the need to remove damage from RNA, and our methodology lends itself well to answer that question. Finally, we are adapting our mutation-analysis technology to probe mutational mechanisms of adducts replicated intrachromosomally in mammalian cells.
This project defines the type, amount and genetic requirements for mutagenesis by carcinogens. It defines the eariy, chemical, steps in the conversion of normal cells into cancer cells. Moreover, the project helps identify the pathways by which cells defend against cancer-causing DNA damage. From the public health perspective, the adducts studied here are candidate biomarkers that could be used to gauge cancer risk.
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