Extended Data Fig. 6: Structure-function and binding studies on the PP domain of MsCAPP.
From: Molecular basis for the initiation of DNA primer synthesis
a. Effect of mutations of MpCAPP100-360 (MpPP) on its polymerase activity. 50 nM MpPP wild-type (WT) (lane 2) or its mutated variants (lanes 3-18) were added to 50 nM DNA substrate (DNA template – oNB1 + FAM-labelled DNA primer – oNB2) and 100 µM dNTPs. The reactions were incubated at 37 °C for 15 min. b. Primase activity of MpPP WT and its mutants. 2 µM MpPP WT protein (lane 2) or its mutants (lanes 3-18) were added to reactions containing 1 µM DNA substrate (oKZ388), 100 µM dNTP mix and 10 µM FAM-labelled GTP (fused via γ-phosphate) (⋆γGTP). The reactions were incubated at 50 °C for 30 min. The products were resolved on 20% urea-PAGE gel. ‘C’ indicates a control reaction without protein. AxA – D177A, D179A, RR – R142A, R143A, KK – K181A, K182A, KQN – K264A, Q265A, N274A. Results are representative of five (panel a) and four (panel b) independent repeats. Oligonucleotide length marker (Nts) is shown on the left of the gel. ‘C’ indicates control without protein. c. Fluorescence polarization assays (FP) reveal that the presence of dinucleotide (rG-dA) does not stimulate binding of MsCAPP111-328 (MsPP) to template. FP: 0–80 µM 5’-3prG-dA-3’ (ATDBio) dinucleotide was added to 5 µM MsPP and 50 nM FAM-DNA (✶ DNA, oKZ409) in presence or absence of 0.1 mM dTTP. d. Stimulation of PP DNA binding affinity in presence of nucleotides is dependent on the template sequence. FP: 0–20 µM MsPP was added to 50 nM ✶ DNA templates (oKZ409, oKZ413, oKZ414 and oKZ416) ± 1 mM GTP and 0.1 mM dATP. e. The efficiency of PP dinucleotide formation is dependent on the −2 base on the template. 1 µM MsPP was added into the reaction containing 1 µM template (lane 2 - oKZ435, lane 3 - oKZ447, lane 4 - oKZ449, lane 5 - oKZ450, lane 6 – oKZ448), 100 µM dATP and 10 µM ✶ γGTP. The reactions were incubated at 50 °C for 30 min. The gel is representative of three independent repeats (left). Signal of synthetized dinucleotides were normalized to signal of dinucleotide in presence of 3’-AAACTAAA-5’ ssDNA template (100%). Data represent the mean ± standard deviation from three independent experiments. Black dots – individual values. ‘C’ indicates control reaction without protein. f. Affinity of PP to template increases with the template length. FP – 0–20 µM MsPP was added to 50 nM ✶ DNA templates (oKZ408–oKZ412). Data representing the mean ± standard deviation from four independent experiments (Panels c, d and f). The mean values were used to calculate the dissociation constants (Kd) shown on the right (Panel d, f); SD – standard deviation of calculated Kd; ND – not determined (Panels d, f).
