Computational protocol: Structures of DNA duplexes containing O6-carboxymethylguanine, a lesion associated with gastrointestinal cancer, reveal a mechanism for inducing pyrimidine transition mutations

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Protocol publication

[…] Initial screenings of crystallization conditions were performed at 277 K by the hanging-drop vapor diffusion method using a kit for nucleic acids reported by Berger et al. (). Two-microliter droplets were equilibrated against 700 μl of the reservoir solution. The optimized conditions for obtaining O6-CMG5T and O6-CMG4C crystals were as follows. For O6-CMG5:T, a droplet of 40 mM sodium cacodylate buffer solution at pH 7.0 containing 1 mM ODN, 10% (v/v) 2-methyl-2,4-pentanediol (MPD), 12 mM spermine tetrahydrochloride, 80 mM sodium chloride, 12 mM potassium chloride, 20 mM magnesium chloride and 1 mM Hoechst33258 (2′-(4-hydroxyphenyl)-6-(4-methyl-1-piperazinyl)-2,6′-bi-1H-benzimidazole) was equilibrated against 35% (v/v) MPD. For O6-CMG4C, a droplet of 40 mM sodium cacodylate buffer solution at pH 7.0 containing 1 mM ODN, 10% (v/v) MPD, 12 mM spermine tetrahydrochloride, 40 mM lithium chloride, 80 mM strontium chloride and 1 mM Hoechst 33258 was equilibrated against 35% (v/v) MPD.O6-CMG5T and O6-CMG4C crystals suitable for X-ray data collections were picked up from their droplets with a nylon loop (Hampton Research) and transferred into liquid nitrogen. X-Ray diffraction experiments of these crystals were performed at 100 K with synchrotron radiation (λ = 1.00 Å at BL-5 A and 0.98 Å at BL-17A) of the Photon Factory in Tsukuba (Japan). Diffracted intensities were recorded on a CCD detector Quantum 315r positioned 200.0 and 155.4 mm from O6-CMG5T and O6-CMG4C crystals, respectively. A total of 180 frames of the patterns for one crystal were taken at 1° oscillation steps with 1 s exposure per frame. Raw diffraction images were indexed, and intensities around Bragg spots were integrated using the computer programs, HKL2000 () for O6-CMG5T data and Mosflm()-Scala () of the CCP4 suite () for O6-CMG4C data. To compensate for the overloaded reflections, the intensity data were merged with those collected at different exposure doses. The crystal data and statistics of data collection are summarized in . [...] Using the program autoMR in the CCP4 suite (), the phases of the two data sets were separately estimated by the molecular replacement method with the unmodified ODN structure d(CGCGAATTCGCG) () as a probe. The atomic parameters were refined using the maximum-likelihood least-squares technique in REFMAC5 () of CCP4 and CNS (). The crystal structures were constructed and modified by adding other molecules and ions using the program Coot () in CCP4. The resultant structures were validated by interpretation of OMIT maps at every nucleotide residue. Electron densities assignable to a magnesium ion and three strontium ions were found in O6-CMG5T and O6-CMG4C, respectively, and these cations were included in the subsequent refinements. The structural restraints applied initially on DNA and Hoechst33258 were released. The Rfactor and Rfree values converged with further rounds of the structure refinements.The statistics of structure refinements are summarized in . The atomic parameters of O6-CMG5T and O6-CMG4C crystal structures were deposited in the protein data bank (PDB-IDs = 4ITD and 4IJ0). shows the 2Fo-Fc electron density maps of the modified nucleotide and its partner in the pair formation, together with the corresponding Fo-Fc omit maps calculated without the pairs. These maps were depicted by the program Dino (). All the residues were traced on the electron density maps. The O6-CMG residues assigned on Fo-Fc omit maps of O6-CMG5T and O6-CMG4C fit well into the 2Fo-Fc maps. All the global and local helical parameters, as well as the torsion angles and pseudo-rotation phase angles of sugar rings, were calculated using the program 3DNA (). Some of them are given in . Figure 2. […]

Pipeline specifications

Software tools iMosflm, CCP4, REFMAC5, CNS, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Diseases Gastrointestinal Neoplasms, Neurofibromatosis 1
Chemicals Cytosine, Bisbenzimidazole, Thymine