Interaction of Formamidopyrimidine-DNA glycosylase (Fpg) with Damaged DNA: Backbone Assignments, Chemical Shift Mapping, and Relaxation Studies.
EMSL Project ID
2442
Abstract
Introduction. Escheria coli formamidopyrimidine-DNA glycosylase (Fpg), a 269-residue, 30.2 kDa metalloprotein, is a trifunctional DNA base excision repair (BER) enzyme that binds double-stranded DNA and performs 3 catalytic activities: (i) DNA glycosylase, (ii) AP lyase, and (iii) deoxyribophosphodiesterase. In the process, Fpg recognizes and removes a wide variety of oxidative DNA lesions with its primary biological substrate the promutatagenic 7,8-dihydro-8-oxoguanine (8-oxoG). Removal of 8-oxoG and other oxidative DNA lesions (2,6-diamino-4-hydroxy-5-formamido pyrimidine, the adenine equivalents, triaminooxazolone, 5-hydroxyuracil, 5-hydroxycytosine, 5,6-dihydrothymine, and a-R-hydroxy-b-ureidoisobutiric acid) is important because cellular DNA is continuously being exposed to endogenous and exogenous agents that damage DNA. Unrepaired, oxidative DNA lesions may have deleterious cellular consequences, including cell death and mutagenesis. In eukaryotes, such damage also plays a role in a number of diseases, including cancer and aging. In order to understand how Fpg performs its many biological activities on DNA in solution, the structure and function of Fpg is being studied by NMR spectroscopy. Using previously requested NMR time on the 800 and 750 MHz (1H) spectrometers we have assigned the majority of the backbone 15N, 1HN, 13Ca, 13Cb, and 13CO resonances of Fpg. A manuscript describing the backbone assignments has recently been accepted for publication in the Journal of Biomolecular NMR and a copy of the accepted manuscript is attached. Having assigned the backbone resonances of Fpg we are now in a position to probe, in detail, the mechanisms of Fpg’s catalytic activities. The primary goals of our current request for time on the 800 and 750 MHz (1H) NMR spectrometers is to: (i) complete the backbone assignments of Fpg bound to a DNA substrate, (ii) complete chemical shift perturbation and cross-saturation studies of Fpg bound to DNA substrate, and (iii) complete the collection of relaxation data for Fpg bound to DNA substrate. SEE HARD COPY FOR FULL TEXT
Project Details
Project type
Capability Research
Start Date
2002-01-30
End Date
2002-11-18
Status
Closed
Released Data Link
Team
Principal Investigator