Cubated with 50 nM PfRecJ at 50 C for 0, two, five, ten, 20 and 30 min (A and D) or 30 min (B and C). Lowercase and uppercase denote RNA and DNA, respectively. (A) Time course of 16nt ssRNA digestion by wt PfRecJ. (B) Identification of essential residues. 3 mutant PfRecJs were made use of to confirm the conserved amino acid residues essential for exonuclease activity. (C) Effect of 30 ribonucleotide on ssRNA digestion by PfRecJ. A 16nt ssRNA with four 30 ribonucleotides (a, u, c and g) was digested by PfRecJ. (D) Impact of ssRNA length on hydrolysis efficiency. ssRNAs with distinctive lengths (12, 16 and 25 nt) have 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 within the 30 0 direction, leaving a hydroxyl group around the shortened 30 finish (Figure 3A). To investigate the possibility that this exonuclease activity was on account of a contaminant protein that copurifies with PfRecJ, we demonstrated that the 30 exonuclease activity on ssRNA was intrinsic to PfRecJ. Many genes from P. furiosus (encoding PCNA,RPA, GINS and primase) had been cloned inside the pDEST17 expression vector, and recombinant proteins have been induced and purified in an identical approach to PfRecJ. None of those recombinant proteins, except RecJ, showed 30 exonuclease activity on ssRNA (data not shown). RecJ protein has several conserved motifs, some of that are also conserved within the DHH superfamily (22,25,27). We mutated 3 conserved motifs to verify their impact on 30 and 50 exonuclease activities on ssRNA and ssDNA,5822 Nucleic Acids Research, 2013, Vol. 41, No.respectively. Altering 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), indicating that the 3 motifs are important to get a potential proofreading function. The 30 exonuclease activity of PfRecJ was identified to depend on the divalent metal ion Mn2, with an optimal concentration selection of 0.1.five mM (Supplementary Figure S2A and B). Other divalent metal ions (Ca2, Cu2, Zn2, Ni2 and Co2) inhibited the 30 exonuclease activity even inside the presence of Mn2 (Supplementary Figure S2C). The biochemical properties of PfRecJ were then characterized applying a 12nt ssRNA (Supplementary Figure S3).Aminoethyl-SS-propionic acid Order PfRecJ exhibited highest activity inside the following situations: pH 7.885588-14-7 Chemscene 0.PMID:25027343 5 (Supplementary Figure S3A), low ion strength (Supplementary Figure S3B) and 70 C reaction temperature (Supplementary Figure S3C). However, the reaction temperature utilized subsequently was 50 C because of the thermal instability of ssRNA at high temperatures (information not shown). The relative activities on ssDNA and ssRNA were comparable (Supplementary Figures S1A and S3D). If PfRecJ features a proofreading function, then the 4 forms of ribonucleotides should be removed having a comparable efficiency. Hence, 4 ssRNAs with several 30 ribonucleotides have been employed to verify the selectivity on the 30 exonuclease activity. Our outcome showed that the four ssRNAs had been digested by PfRecJ in almost equalefficiency (Figure 3C), indicating that PfRecJ has no clear selectivity on the 30 ribonucleotide. The RNA primer synthesized by primase is 120 nt lengthy (11,40). Hence, we investigated the cleavage efficiency of PfRecJ on ssRNAs with unique lengths. The outcomes showed that the cleavage efficiencies on ssRNAs with different lengths had been comparable, with some preferen.