PMC

A) Representative 8%PAGE/ 1X TAE/ 0.1% SDS gel panels of annealing timecourses comparing spontaneous annealing (no protein) to reactions containing 288 nM of the N-terminal domain of TelA (TelA (1–106)). The migration positions of the 5’-³²P endlabeled reporter oligonucleotide (ss) and the duplex product (ds) are indicated. B) A plot of annealing rate vs. TelA concentration is shown comparing the annealing rates of wildtype TelA (blue) and TelA (1–106) (red). The wildtype TelA data ranging from the spontaneous rate of annealing to 154 nM of TelA was previously shown in . Data for annealing rates with higher concentrations of wt TelA (192 nM– 385 nM) were collected in tandem with TelA (1–106) annealing rates and have not been previously shown. The mean and standard deviation are shown and are derived from three independent experiments.

Annealing schedule of forward and reverse annealing. The red and blue lines indicate the chronological changes in annealing parameter s in forward and reverse annealing, respectively.

Results of the control experiments with sample B, which has NM electrodes made of Au/Ta. L_ch = 2.75 µm, t = 100 nm for sample B. The widths of F1 and F2 were 0.5 and 2.0 µm, respectively. (a) The spin accumulation signals of sample B measured by the NL4T method. Black, red, blue and green show the measured V_nl before annealing after annealing at 300 °C, 350 °C and 400 °C, respectively. The inset shows the result of the L3T measurement after annealing at 400 °C. (b) Results of the Hanle measurements of sample B. Black, red and blue show the measured V_nl before annealing after annealing at 300 °C and 350 °C, respectively. (c) I–V characteristics of sample B. Black, red, blue and green show the measured V_nl before annealing after annealing at 300 °C, 350 °C and 400 °C, respectively. The inset shows the RA product normalized by the value before annealing measured at 1.0 mA.

Pif1-catalyzed strand annealing using longer DNA substrates. (a) Schematic illustration for strand annealing experiments using 80nt strand and 80nt CS to generate an 80bp blunt end dsDNA product. (b) Results for Pif1 (200 nM) annealing of the 80nt strand (2.6 nM) with radiolabeled 80nt CS (2 nM) to generate an 80bp blunt DNA product in the presence of ATP and MgCl₂ (red circles). Annealing by Pif1 in the absence of ATP but in the presence of MgCl₂ is shown as green squares. Spontaneous annealing in the absence of enzyme in the presence of ATP and MgCl₂ was also measured (blue diamonds). Data were fit to a simple annealing mechanism (Scheme ) to obtain second-order rate constants for annealing of 3.0 × 10⁶ M^–1 s^–1 in the presence of Pif1 and ATP, 2.9 × 10⁷ M^–1 s^–1 in the presence of Pif1 but in the absence of ATP, and 3.0 × 10⁵ M^–1 s^–1 in the absence of Pif1.