DNA is constantly exposed to endogenous and exogenous agents that cleave the biopolymer and produce lesions by modifying its nucleobases. The presence of these lesions in DNA in vivo is associated with aging, diseases such as cancer, and other genetically based diseases. The overall goal of the proposed research is to probe the effects of the formamidopyrimidines (Fapy), an important family of nucleic acid lesions, on the structure and function of DNA. Fapy lesions are produced from the purines (dA, dG) by a variety of oxidative stress mechanisms. Despite their importance, the effects of the Fapy lesions on the function of DNA at the molecular level are not well understood. We recently reported the first chemical synthesis of oligonucleotides containing these lesions and their C-nucleoside analogues. In vitro experiments involving polymerase and repair enzymes by us have uncovered very interesting, potentially biologically important properties of the Fapy lesions. For instance, we have observed similarities and differences to the closely related 8-oxopurines in how the Fapy lesions interact with the Fpg repair protein. We have a so detected that both Fapy lesions decrease the fidelity of the DNA polymerase Klenow exo-, resulting in an increased level of dA misincorporation. In unpublished in vivo experiments we have shown that Fapy*dG is significantly more mutagenic in E. coli than OxodG is in the same assay. During the next funding period we request funds to continue in vitro and in vivo studies of the effects of the Fapy lesions on DNA polymerase and repair enzymes. New research directions include studying the effects of the nucleotide triphosphates of the formamidopyrimidines on polymerase and repair enzymes, and the development of fluorescent based sensors, which will detect these lesions at specific sites in DNA Increased understanding of the effects of the Fapy lesions on nucleic acid structure and function will be useful for understanding the association between nucleic acid damage and aging, as well as the etiology of diseases such as cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA074954-09
Application #
7072280
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Okano, Paul
Project Start
1997-09-10
Project End
2008-06-30
Budget Start
2006-07-01
Budget End
2008-06-30
Support Year
9
Fiscal Year
2006
Total Cost
$271,994
Indirect Cost
Name
Johns Hopkins University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Pande, Paritosh; Haraguchi, Kazuhiro; Jiang, Yu-Lin et al. (2015) Unlike catalyzing error-free bypass of 8-oxodGuo, DNA polymerase ? is responsible for a significant part of Fapy·dG-induced G ? T mutations in human cells. Biochemistry 54:1859-62
Zhang, Bintian; Guo, Liang-Hong; Greenberg, Marc M (2012) Quantification of 8-oxodGuo lesions in double-stranded DNA using a photoelectrochemical DNA sensor. Anal Chem 84:6048-53
Greenberg, Marc M (2012) The formamidopyrimidines: purine lesions formed in competition with 8-oxopurines from oxidative stress. Acc Chem Res 45:588-97
Krishnamurthy, Nirmala; Haraguchi, Kazuhiro; Greenberg, Marc M et al. (2008) Efficient removal of formamidopyrimidines by 8-oxoguanine glycosylases. Biochemistry 47:1043-50
Weledji, Yvonne N; Wiederholt, Carissa J; Delaney, Michael O et al. (2008) DNA polymerase bypass in vitro and in E. coli of a C-nucleotide analogue of Fapy-dG. Bioorg Med Chem 16:4029-34
Xue, Liang; Greenberg, Marc M (2007) Facile quantification of lesions derived from 2'-deoxyguanosine in DNA. J Am Chem Soc 129:7010-1
Patro, Jennifer N; Wiederholt, Carissa J; Jiang, Yu Lin et al. (2007) Studies on the replication of the ring opened formamidopyrimidine, Fapy.dG in Escherichia coli. Biochemistry 46:10202-12
Das, Aditi; Boldogh, Istvan; Lee, Jae Wan et al. (2007) The human Werner syndrome protein stimulates repair of oxidative DNA base damage by the DNA glycosylase NEIL1. J Biol Chem 282:26591-602
Kalam, M Abul; Haraguchi, Kazuhiro; Chandani, Sushil et al. (2006) Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells. Nucleic Acids Res 34:2305-15
Imoto, Shuhei; Patro, Jennifer N; Jiang, Yu Lin et al. (2006) Synthesis, DNA polymerase incorporation, and enzymatic phosphate hydrolysis of formamidopyrimidine nucleoside triphosphates. J Am Chem Soc 128:14606-11

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