Computational protocol: Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

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Protocol publication

[…] D135A AlkB X-ray diffraction data were collected at 100 K on a Rigaku MicroMax-007 rotating Cu anode generator with a Saturn-92 CCD detector. Wt AlkB X-ray diffraction data were collected on beamline X29A at the National Synchrotron Light Source (NSLS), Brookhaven National Labs. All data were integrated and scaled using the d*TREK program suite . All structures were phased by molecular replacement using Phaser with the AlkB structure (pdbid: 2FD8) as the search model. The models were built using the program Coot , and refinement was carried out using the programs CNS , and REFMAC5 , with TLS refinement , . All data collection and refinement statistics are found in . All molecular graphics figures were prepared using PyMOL . Coordinates for the structures of the unliganded AlkB (pdbid: 3KHB) and the AlkB-ssDNA complex containing 1-meG (pdbid: 3KHC) have been deposited in the Protein Data Bank (PDB). [...] AlkB catalytic activity was assayed using a 15-mer oligonucleotide containing a single 1-methyladenine (1-meA) base. Reactions contained 20 mM Tris 8.0, 6 mM Fe(NH4)2(SO4)2, 1 mM 2-OG, 10 mM ascorbate, 10 µM DNA (ChemGene) substrate, and 500 nM AlkB wild type or mutant enzymes. Reactions (10 µl) were carried out at 37°C for 10 minutes and quenched with 50 mM EDTA at 2.5 minute time points. Samples were prepared for matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis using a C18 Zip-tip (Millipore) according to the manufacturer's protocols. Samples were mixed at a 1∶1 ratio with a 3-hydroxypicolinic acid matrix (10∶1 ratio of 3-hydroxypicolinic acid 50 mg/ml to 0.1 M diammonium hydrogen citrate) and spotted on a Bruker Daltonics MALDI-TOF MS MTP target frame III. Samples were analyzed in the negative ion mode on a Bruker Daltonics instrument. Peaks corresponding to the damaged substrate (4527 Da) and undamaged substrate (4512 Da) for the 1-methyladenine oligonucleotide were integrated using the area below the curve algorithm in SigmaPlot to determine the percent of the substrate repaired. 1-methylguanine (1-meG) activity assays were carried out under the same conditions outlined above with a 17-nucleotide 1-meG (ChemGene). Protein concentration was increased to 1 µM and reactions were carried out for 25 minutes. Peaks corresponding to the damaged substrate (5176 Da) and undamaged substrate (5162 Da) were integrated in the same manner as the 1-meA experiments. Percent of repaired substrate was converted to µM by multiplying by the total substrate in the reaction and plotted as µM vs. time to obtain a rate in the form µM/min. All experiments were carried out in triplicate and the data averaged. […]

Pipeline specifications

Software tools Coot, CNS, REFMAC5, PyMOL, SigmaPlot
Applications Miscellaneous, Small-angle scattering, Protein structure analysis
Organisms Escherichia coli
Chemicals Purines, Pyrimidines