Dcmpnpp

To obtain the binary complex of Asn279Ala polβ-DNA complex, polβ was incubated with a single-nucleotide gapped DNA containing a 16-mer template (5′-CCGAC[G or O6-methylguanine]GCGCATCAGC-3′), a complementary 10-mer upstream primer (5′-GCTGATGCGC-3′), and a 5-mer downstream primer (5′-pGTCGG-3′). Subsequently, a 10-fold molar excess of nonhydrolyzable dTMPNPP or dCMPNPP (Jena Bioscience) was added to the binary complex. Ternary Asn279Ala polβ-DNA complex co-crystals with nonhydrolyzable dNTP analogs paired with templating G or O6-methylguanine (O6MeG) were grown in a buffer solution containing 50 mM imidazole, pH 7.5, 14–23% PEG3400, and 350 mM sodium acetate as described previously [19 (link)]. The diffraction data were collected at 100 K at the beamline 5.0.3 at the Advanced Light Source, Lawrence Berkeley National Laboratory. All diffraction data were processed using HKL2000. The structures were solved by molecular replacement using a ternary complex structure with a closed conformation (PDB ID 1BPY) as the search model [22 (link)]. The model was built using COOT and refined using Phoenix, and the figures were generated using PyMOL.

The ternary polη complex with the templating NHMG was prepared and crystallized with slight modification of published protocols [34 (link), 39 ]. Purified polη (9 mg/ml) was mixed with a 1.2 molar excess of duplex DNA containing NHMG lesion (primer, 5´-AGTGTGAG-3´ and template, 5´-CAT(NHMG)CTCACACT-3´). Subsequently, a 10-fold molar excess of nonhydrolyzable dCMPNPP (Jena Bioscience) was added to the binary complex of the polη:NHMG duplex DNA. The ternary complex co-crystals with dCMPNPP paired with the templating NHMG were obtained using the hanging drop method in a buffer solution containing 100 mM MES pH 6.0–6.5 and 18% PEG2000 MME. Crystals were harvested and cryoprotected in mother liquor supplemented with 20% glycerol and were flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K at the beamline 19-ID at the Advanced Photon Source, Argonne National Laboratory. The diffraction data were processed using HKL2000 and the scaled set was converted to structure factors by CCP4 [40 (link)]. Structures were solved by molecular replacement using polη-dG:dCTP* structure (PDB ID: 4O3N) as a search model. The model was built using COOT [41 (link)]and refined using PHENIX [42 (link)]. MolProbity in the PHENIX GUI was used to generate Ramachandran plots [43 (link)]. All the crystallographic figures were generated using PyMOL.