Yzed RNA at relative low price, we confirmed its possible to proofread the 30 mismatched RNA primer by comparing its kinetic parameters with these of primase and PolB. The kinetics of P. furiosus primase, PolB and RecJ had been calculated by doublereciprocal plotting (Table three). The Kcat/Km with the mismatched RNA/DNA hybrid (in the presence/absence of RPA) was 3fold greater than that on the matched hybrid. Nevertheless, the decrease Kcat/Km with the matched RNA/DNA hybrid was comparable with that of primase and PolB. For primase and PolB, the Kcat/Km of the mismatched RNA/DNA hybrid can’t be determined accurately because of the incredibly low reaction rate (Supplementary Figure S5). These final results indicate that PfRecJ can efficiently eliminate the 30 mismatched ribonucleotide, but would not block the incorporation of a matched NMP and dNMP by primase and PolB, respectively. Furthermore, the presence of a 30 mismatched ribonucleotide totally blocks the incorporation from the next nucleoside phosphate by PolB (Supplementary Figure S5), indicating that the removal from the 30 mismatched ribonucleotide is crucial for efficient DNA synthesis. Our findings that Thermus thermophilus RecJ preferably binds ssDNA (Supplementary Figure S6A), and that PfRecJ preferentially binds ssRNA compared with RNA/DNA hybrids (Supplementary Figure S6B and C), are consistent with the higher 30 exonuclease activity of PfRecJ on ssRNA. RecJ proofreads 30 mismatched ribonucleotides within the DNA elongation reaction of PolB Neither wt nor 30 exonuclease eficient PolB can effectively extend an RNA primer if the 30 ribonucleotide is mismatched (Figure 5A, lanes two and three). On incubation with PfRecJ, the two types of PolB can extend theTable three. Comparison of Km and Kcat of P. furiosus RecJ, primase and PolBEnzymes Substrates Km (mM) Pol B Primase RecJc 3 matched RNA/DNA 30 mismatched RNA/DNA 30 matched RNA/DNA 30 mismatched RNA/DNA ssRNA 30 mismatched RNA/DNA 30 matched RNA/DNA ssRNA 30 mismatched RNA/DNA 30 matched RNA/DNAaKinetic parameter Kcat (min) 0.00034 ND 0.017 ND 0.24 0.040 0.015 0.34 0.12 0.037 Kcat/Km (min mM)0.0080 NDb 0.14 ND 0.48 0.62 0.70 0.73 0.29 0.0.042 ND 0.12 ND 0.50 0.065 0.022 0.47 0.42 0.RecJdKm and Kcat have been calculated by doublereciprocal plotting applying the initial reaction rates of ssRNA as well as matched (g/C) and mismatched (g/G) RNA/DNA hybrids at numerous substrate concentrations (0.1, 0.two, 0.5, 1, two.five and ten mM). a To examine the catalytic efficiency of each and every enzyme for different substrates, various substrates (such as ssRNA, 30 matched RNA/DNA hybrids, or mismatched RNA/DNA hybrids) had been used to calculate the kinetic parameter of each enzyme.Price of Boc-NH-PEG11-NH2 b The kinetic parameters cannot be calculated accurately because the extension efficiency is also low to generate a clear solution (Supplementary Figure S5).(Dtpby)NiBr2 Order c The kinetic parameters were determined in the absence of RPA.PMID:24733396 d The kinetic parameters were determined in the presence of 1 mM RPA. ND, not determined.30 mismatched RNA primer into extended fragments (Figure 5A, lanes 11 and 12). The wt PolB also has 30 0 exonuclease activity on ssRNA (5 on the activity of RecJ, Supplementary Figure S7). Nevertheless, it couldn’t effectively proofread the 30 mismatched RNA primer (Figure 5A, lane 2). After the 30 mismatched RNA/DNA hybrid was incubated with PfRecJ, the yield of fragments extended by primase also enhanced (Figure 5A, lanes 2 and ten). Incubation with RecJ decreased the extension yield from the 30 mismatched RNA primer compared using the.
