Computational protocol: Recognition and processing of double-stranded DNA by ExoX, a distributive 3′–5′ exonuclease

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Protocol publication

[…] Oligonucleotides were purchased from Augct Bio Ltd. (Beijing, China). For the 3′ overhanging dsDNA substrate, two 17-mer oligonucleotides (5′-CGGATCCACAATGACCT-3′ and 5′-GTCATTGTGGATCCGAG-3′) were annealed to create a 17 bp dsDNA with a 3′ dinucleotide overhang. For the blunt-ended dsDNA substrate, two 12-mer oligonucleotides (5′-CTCGAATCTACA-3′ and 5′-TGTAGATTCGAG-3′) were annealed to generate a 12 bp blunt-ended dsDNA. For the 3′ recessed mismatch-containing dsDNA substrate, the same 17-mer oligonucleotide from one strand of the 3′overhanging dsDNA (5′-GTCATTGTGGATCCGAG-3′) was self-annealed to form a DNA duplex with a 5 nt 5′ overhang and mispaired 3′ terminal nucleotides.For crystallization, ExoX was incubated with dsDNA at a ratio of 1:0.6 for 30 min at 4°C, and the resultant protein–DNA complexes were purified using Superdex 200 gel filtration columns. For crystallization of the Se-Met-derivative ExoX in complex with 3′ overhanging dsDNA (complex I), purified complexes were concentrated to 2 mg/ml. ExoX in complex with blunt-ended dsDNA (complex II) and ExoX in complex with 3′ recessed mismatch-containing dsDNA (complex III) were concentrated to 1 mg/ml. All crystals were grown via the hanging-drop vapor diffusion method at 16°C using 0.1 M Tris–HCl (pH 8.0), 0.2 M NaCl and 20% PEG 3350 for complex I and 0.1 M MES (pH 6.3) and 17% PEG 6000 for complexes II and III. Individual rod-shaped crystals of all three complexes were obtained through microseeding.Before data collection, crystals were transferred to a reservoir solution containing 25% PEG 3350 and 20% sucrose and flash frozen in liquid nitrogen. All radiographic data sets were collected on beamline BL17U at the Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China) and processed using HKL2000 (). For the Se-Met derivative complex I, the phase was determined via single-wavelength anomalous dispersion. An electron density map and initial model were built using PHENIX (). Further model building was performed with COOT (), and refinement was conducted using REFMAC () and PHENIX-Refine (). For complexes II and III, the structures were solved via molecular replacement and refined using REFMAC and PHENIX-Refine. Data collection and refinement statistics are presented in Supplementary Table S1. […]

Pipeline specifications

Software tools PHENIX, Coot
Application Protein structure analysis
Organisms Escherichia coli