The goals of this proposal are to continue the development of a general method for the preparation 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 at attempt to relate these structural changes to the mutations that they induce. The data generated in these studies will be used to test the hypothesis that the type of mutation induced is dependent on the adduct structure and the DNA sequence within which it is located. We will also attempt to specifically relate this analysis to the question of the importance of non-targeted or semi-targeted mutagenesis. In addition, these site- specifically modified templates will also be used to characterize the stable complex which we have observed to form between a DNA lesion and a DNA helicase. This proposal is focussed on two important and well- studied classes of carcinogenic DNA adducts: aromatic amines and polycyclic aromatic hydrocarbons. However, our long-term objective is to develop techniques to place any DNA lesion into a specific DNA sequence, even including those where the chemistry needed to prepare the lesion is not now known or where the instability of the lesion to currently available oligonucleotide synthesis technology makes the synthesis prohibitive. The relevance of this work towards the progress of understanding the initiation of cancer in eukaryotic cells is two- fold. First, studying the relationship between adduct structure within a specific sequence context and induced mutations and mutation frequency will lead to an understanding of the parameters which define a mutational hotspot, many of which are thought to be important in oncogene activation. Second, studying the irreversible binding of a protein to a DNA adduct site may clarify the role these structures might play in the induction of chromosomal breaks or rearrangements.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA040605-08
Application #
2090283
Study Section
Metabolic Pathology Study Section (MEP)
Project Start
1986-05-01
Project End
1996-01-31
Budget Start
1994-02-01
Budget End
1995-01-31
Support Year
8
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Wayne State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
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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