Work

NH2-terminal Proline Acts as a Nucleophile in the Glycosylase/AP-Lyase Reaction Catalyzed by Escherichia coli Formamidopyrimidine-DNA Glycosylase (Fpg) Protein

Year: 1997

Type: article

Abstract: Formamidopyrimidine-DNA glycosylase (Fpg) protein plays a prominent role in the repair of oxidatively damaged DNA in Escherichia coli. The protein possesses three enzymatic activities, hydrolysis of the N-glycosidic bond (DNA glycosylase), beta-elimination (AP lyase), and delta-elimination; these functions act in a concerted manner to excise oxidized deoxynucleosides from duplex DNA. Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A. P. (1995) J. Biol. Chem. 270, 11671-11677); this protein-DNA complex can be trapped by reduction with sodium borohydride. By digesting the stable, covalently linked intermediate with proteases and determining the accurate mass of the products by negative electrospray ionization-mass spectrometry, we show that the N-terminal proline of Fpg protein is linked to DNA and, therefore, is identified as the nucleophile that initiates the catalytic excision of oxidized bases from DNA. This experimental approach may be applicable to the analysis of other protein-DNA complexes.

Source: Journal of Biological Chemistry

DOI: 10.1074/jbc.272.8.5335

Authors: Dmitry O. Zharkov, Robert A. Rieger, Charles R. Iden, Arthur P. Grollman

Institution: Stony Brook University

Language: English

Topic: DNA Repair Mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $3,430

Funders: National Cancer Institute, National Institute of Environmental Health Sciences