Computational protocol: Structure and mechanism of human PrimPol, a DNA polymerase with primase activity

Similar protocols

Protocol publication

[…] Initial crystallization conditions for the native and SeMet-labeled PrimPol (residues 1 to 354)–DNA–dNTP ternary complexes were determined with matrix screens (Hampton Research and Qiagen). Template-primer DNAs were annealed and mixed with PrimPol in a 1.2:1 molar ratio to a final concentration of 0.3 mM in 25 mM tris (pH 7.5), 155 mM NaCl, 1.5 mM TCEP, 12 mM MgCl2, and 7 mM dATP or nonhydrolyzable dApNHpp analog (Jena Bioscience). The complexes were incubated at room temperature for 5 min and then centrifuged at 10,000 rpm for 10 min at 4°C. The crystals were grown at 20°C by a hanging drop method against a reservoir solution containing 200 mM CaCl2 (for native PrimPol) or 300 mM NaSCN (for SeMet-labeled proteins) and 17 to 23% PEG 3350. The rod-like crystals appeared in 1 to 3 weeks; microseeding was required in some conditions to produce crystals up to ~0.2 mm in length. For data collection, crystals were cryoprotected stepwise in reservoir solution supplemented with 24% PEG 3350 and 10% and then 20% glycerol and flash-frozen in liquid nitrogen.X-ray data on the native and SeMet-labeled PrimPol-DNA-dNTP cocrystals were measured at the Advanced Photon Source in Chicago (beamlines 24-ID and 23-ID). The data sets were processed using XDS (), iMosflm (), and aimless and pointless in the CCP4 program package (). The native crystals diffracted to ~2.2 Å resolution and belonged to space group P1, whereas the SeMet crystals diffracted to ~3 to 3.5 Å resolution and belonged to space groups C2 and P21 (table S1). The P1 and P21 crystals contained two PrimPol-DNA-dNTP ternary complexes in the crystallographic asymmetric unit, whereas the C2 crystals contained only one complex. To calculate experimental phases for structure determination, we used SAD data measured at Se-K absorption edge (~0.9791 Å) on both forms of SeMet crystals (C2 and P21). The phases were calculated with program SHARP (), and the density-modified, noncrystallographic symmetry–averaged maps were calculated using the program DM (). The experimental maps showed reasonably clear electron density for most regions of the protein-DNA complexes. A model was built manually in Coot (), refined in Phenix (), and then positioned in the native P1 space group by molecular replacement. The model was further built in Coot and refined in Phenix in an iterative manner (to the 2.2 Å diffraction limit of the native data). As an additional aid to model building, we labeled the template strand with 5-iododeoxyuracil and measured another datum on these crystals at low energy (~7 keV or ~1.77 Å). The location of iodine peaks in the anomalous difference map calculated using anomalous data from these iodinated DNA crystals unambiguously confirmed the register of both DNA and primer strands in our model. The final model was refined to ~2.2 Å resolution with Rfree and Rwork values of ~25.4 and 20.9%, respectively, and displayed good stereochemistry (table S1). The two ternary complexes (A and B) in the crystallographic asymmetric unit of native space group P1 were very similar in structure, except for residues 1 to 17, which could be traced as an α helix in complex A but not in complex B. We considered whether this α helix in complex A was from a symmetry-related molecule (residues 244 to 260), but the orientation in that case would be reversed and did not match the electron density. Two other regions (residues 18 to 34 and 201 to 259) could not be traced in either complex A or complex B. These disordered regions lacked any predictable secondary structure and might only become ordered upon interaction with other protein partners. […]

Pipeline specifications