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Rapid determination of the active fraction of DNA repair glycosylases: a novel fluorescence assay for trapped intermediates.

ABSTRACT: Current methods to measure the fraction of active glycosylase molecules in a given enzyme preparation are slow and cumbersome. Here we report a novel assay for rapidly determining the active fraction based on molecular accessibility of a fluorescent DNA minor groove binder, 4',6-diamidino-2-phenylindole (DAPI). Several 5,6-dihydrouracil-containing (DHU) DNA substrates were designed with sequence-dependent DAPI-binding sites to which base excision repair glycosylases were covalently trapped by reduction. Trapped complexes impeded the association of DAPI in a manner dependent on the enzyme used and the location of the DAPI-binding site in relation to the lesion. Of the sequences tested, one was shown to give an accurate measure of the fraction of active molecules for each enzyme tested from both the Fpg/Nei family and HhH-GPD Nth superfamily of DNA glycosylases. The validity of the approach was demonstrated by direct comparison with current gel-based methods. Additionally, the results are supported by in silico modeling based on available crystal structures.

SUBMITTER: Blaisdell JO

PROVIDER: S-EPMC1865064 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Rapid determination of the active fraction of DNA repair glycosylases: a novel fluorescence assay for trapped intermediates.

Blaisdell Jeffrey O JO Wallace Susan S SS

Nucleic acids research 20070208 5

Current methods to measure the fraction of active glycosylase molecules in a given enzyme preparation are slow and cumbersome. Here we report a novel assay for rapidly determining the active fraction based on molecular accessibility of a fluorescent DNA minor groove binder, 4',6-diamidino-2-phenylindole (DAPI). Several 5,6-dihydrouracil-containing (DHU) DNA substrates were designed with sequence-dependent DAPI-binding sites to which base excision repair glycosylases were covalently trapped by re ...[more]

PMID: 17289752

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Project description:In the present work, the conformational dynamics and folding pathways of i-motif DNA were studied in solution and in the gas-phase as a function of the solution pH conditions using circular dichroism (CD), photoacoustic calorimetry analysis (PAC), trapped ion mobility spectrometry-mass spectrometry (TIMS-MS), and molecular dynamics (MD). Solution studies showed at thermodynamic equilibrium the existence of a two-state folding mechanism, whereas during the pH = 7.0 → 4.5 transition a fast and slow phase (ΔHfast + ΔHslow = 43 ± 7 kcal mol-1) with a volume change associated with the formation of hemiprotonated cytosine base pairs and concomitant collapse of the i-motif oligonucleotide into a compact conformation were observed. TIMS-MS experiments showed that gas-phase, kinetically trapped i-motif DNA intermediates produced by nanoESI are preserved, with relative abundances depending on the solution pH conditions. In particular, a folded i-motif DNA structure was observed in nanoESI-TIMS-MS for low charge states in both positive and negative ion mode (e.g., z = ±3 to ±5) at low pH conditions. As solution pH increases, the cytosine neutralization leads to the loss of cytosine-cytosine+ (C·CH+) base pairing in the CCC strands and in those conditions we observe partially unfolded i-motif DNA conformations in nanoESI-TIMS-MS for higher charge states (e.g., z = -6 to -9). Collisional induced activation prior to TIMS-MS showed the existence of multiple local free energy minima, associated with the i-motif DNA unfolding at z = -6 charge state. For the first time, candidate gas-phase structures are proposed based on mobility measurements of the i-motif DNA unfolding pathway. Moreover, the inspection of partially unfolded i-motif DNA structures (z = -7 and z = -8 charge states) showed that the presence of inner cations may or may not induce conformational changes in the gas-phase. For example, incorporation of ammonium adducts does not lead to major conformational changes while sodium adducts may lead to the formation of sodium mediated bonds between two negatively charged sides inducing the stabilization towards more compact structures in new local, free energy minima in the gas-phase.

Dataset Information

Rapid determination of the active fraction of DNA repair glycosylases: a novel fluorescence assay for trapped intermediates.

Publications

Rapid determination of the active fraction of DNA repair glycosylases: a novel fluorescence assay for trapped intermediates.

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