Perfectly matched solution.Figure 1. Incorporation fidelity of P. furiosus primase. The extension fidelity of an RNA primer by P. furiosus primase was determined in a buffer consisting of 40 mM HEPES (pH six.4), 30 mM NaCl, ten mM MnCl2 and 4 U RNase inhibitor (Rnsin). P. furiosus primase (100 nM) was incubated with 100 nM recessed RNA primer NA template substrate at 50 C for two, four and 8 min. 4 types of NTP (50 mM) had been added singly to every single reaction to establish the fidelity of the initially incorporated ribonucleotide. The corresponding template bases of each and every primer emplate substrate are listed at the bottom in the image.Nucleic Acids Study, 2013, Vol. 41, No. 11Figure two. Extension of RNA primer by P. furiosus loved ones B DNA polymerase. The extension of RNA primers annealed to complementary DNA templates was determined within a buffer consisting of 20 mM Tris Cl, pH 8.8, ten mM (NH4)2SO4, 10 mM KCl, two mM MgSO4, 0.1 Triton X-100, one hundred ng/ml BSA, 100 mM dNTPs and 4 U Rnsin. About 50 nM of RNA primer NA template substrates was incubated with one hundred nM Pfu DNA polymerase at 50 C for 0, 1, 2, 4, eight, 15, 30 and 60 min (A) or 30 min (B and C). Various RNA primers and DNA templates had been annealed to kind the matched and mismatched RNA/DNA hybrids applied in the extension reactions. Lowercase and uppercase denote RNA and DNA, respectively.Figure 3. Biochemical characterization of P. furiosus RecJ on RNA substrates. The 30 0 exonuclease activity of PfRecJ on ssRNA was determined within a buffer consisting of 20 mM Tris Cl (pH 7.five), 30 mM NaCl, ten mM KCl, five mM DTT, 0.25 mM MnCl2, one hundred ng/ml BSA and four U Rnsin. Substrates (50 nM) had been incubated with 50 nM PfRecJ at 50 C for 0, two, 5, 10, 20 and 30 min (A and D) or 30 min (B and C). Lowercase and uppercase denote RNA and DNA, respectively.X-alpha-Gal (A) Time course of 16-nt ssRNA digestion by wt PfRecJ.Piracetam (B) Identification of key residues.PMID:23522542 Three mutant PfRecJs were applied to confirm the conserved amino acid residues important for exonuclease activity. (C) Impact of 30 ribonucleotide on ssRNA digestion by PfRecJ. A 16-nt ssRNA with four 30 ribonucleotides (a, u, c and g) was digested by PfRecJ. (D) Impact of ssRNA length on hydrolysis efficiency. ssRNAs with distinct lengths (12, 16 and 25 nt) had been digested by PfRecJ.Therefore, we characterized the activity of PfRecJ on ssRNA and ssDNA. In contrast to its activity on ssDNA (Supplementary Figure S1A), PfRecJ hydrolyzed ssRNA in the 30 0 direction, leaving a hydroxyl group around the shortened 30 end (Figure 3A). To investigate the possibility that this exonuclease activity was because of a contaminant protein that co-purifies with PfRecJ, we demonstrated that the 30 exonuclease activity on ssRNA was intrinsic to PfRecJ. Quite a few genes from P. furiosus (encoding PCNA,RPA, GINS and primase) were cloned inside the pDEST17 expression vector, and recombinant proteins were induced and purified in an identical strategy to PfRecJ. None of these recombinant proteins, except RecJ, showed 30 exonuclease activity on ssRNA (information not shown). RecJ protein has a number of conserved motifs, some of which are also conserved within the DHH superfamily (22,25,27). We mutated three conserved motifs to verify their impact on 30 and 50 exonuclease activities on ssRNA and ssDNA,5822 Nucleic Acids Study, 2013, Vol. 41, No.respectively. Changing D34, D36 and D83 to alanine and replacing the 437GGGH440 motif with 437LLLA440 resulted in loss of each 30 and 50 exonuclease activities (Figure 3B and Supplementary Figure S1B), ind.
