The goals of this proposal are to develop improved methods to prepare and purify large amounts of oligodeoxyribonucleotides containing chemically well-defined damage at unique and specific locations, to use these molecules to determine how specific adducts affect the three-dimensional structure of a DNA duplex, and to attempt to relate these structural changes to the mutations and biochemical effects that they induce. The data generated in these studies will be used to test the hypothesis that the type of mutation and other biochemical effects induced by DNA adducts are dependent on the adduct structure and the DNA sequence within which it is located. Retrospective analysis of these biological consequences are expected to provide an understanding of the molecular mechanism of mutagenesis. To reach these goals, Dr. Romano plans to use primarily multidimensional NMR, but also circular dichroism and chemical methods, to elucidate the three-dimensional structure of DNA modified with acetylaminofluorene (AAF), aminofluorene (AF), and benzo(a)pyrene (BaP) adducts. These DNA adducts represent relevant and well-studied examples of chemical carcinogens. He also plans to use physical and enzymatic techniques to characterize the interactions between a DNA polymerase and/or a DNA helicase and DNA containing these DNA adducts.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Metabolic Pathology Study Section (MEP)
Program Officer
Okano, Paul
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Wayne State University
Schools of Arts and Sciences
United States
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Liyanage, Pramodha S; Walker, Alice R; Brenlla, Alfonso et al. (2017) Bulky Lesion Bypass Requires Dpo4 Binding in Distinct Conformations. Sci Rep 7:17383
Brenlla, Alfonso; Rueda, David; Romano, Louis J (2015) Mechanism of aromatic amine carcinogen bypass by the Y-family polymerase, Dpo4. Nucleic Acids Res 43:9918-27
Brenlla, Alfonso; Markiewicz, Radoslaw P; Rueda, David et al. (2014) Nucleotide selection by the Y-family DNA polymerase Dpo4 involves template translocation and misalignment. Nucleic Acids Res 42:2555-63
Vrtis, Kyle B; Markiewicz, Radoslaw P; Romano, Louis J et al. (2013) Carcinogenic adducts induce distinct DNA polymerase binding orientations. Nucleic Acids Res 41:7843-53
Markiewicz, Radoslaw P; Vrtis, Kyle B; Rueda, David et al. (2012) Single-molecule microscopy reveals new insights into nucleotide selection by DNA polymerase I. Nucleic Acids Res 40:7975-84
Federley, Richard G; Romano, Louis J (2010) DNA polymerase: structural homology, conformational dynamics, and the effects of carcinogenic DNA adducts. J Nucleic Acids 2010:
Vooradi, Venkataramana; Romano, Louis J (2009) Effect of N-2-acetylaminofluorene and 2-aminofluorene adducts on DNA binding and synthesis by yeast DNA polymerase eta. Biochemistry 48:4209-16
Christian, Thomas D; Romano, Louis J; Rueda, David (2009) Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution. Proc Natl Acad Sci U S A 106:21109-14
Christian, Thomas D; Romano, Louis J (2009) Monitoring the conformation of benzo[a]pyrene adducts in the polymerase active site using fluorescence resonance energy transfer. Biochemistry 48:5382-8
Lone, Samer; Romano, Louis J (2007) The role of specific amino acid residues in the active site of Escherichia coli DNA polymerase I on translesion DNA synthesis across from and past an N-2-aminofluorene adduct. Biochemistry 46:2599-607

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