Journal: Nature Communications

Article Title: CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation

doi: 10.1038/s41467-021-23535-9

Figure Lengend Snippet: a MpCAPP needs both NTPs and dNTPs for primer synthesis. In all, 4 µM MpCAPP wild-type protein was added into 10 ng/µl circular M13 ssDNA substrate in presence of 2.5 µM dNTPs (FAM-labelled dUTP) + 2.5–100 µM non-labelled NTPs and MpPrimBuffer. The reaction was incubated at 50 °C for 30 min and the products were resolved by denaturing PAGE. b MpCAPP FAM-labelled UTP incorporation is very poor. In total, 4 µM MpCAPP was added into 10 ng/µl circular M13 ssDNA substrate in presence of 2.5 µM NTPs (FAM-labelled UTP) + 2.5–100 µM non-labelled dNTPs and MpPrimBuffer. The reaction was incubated at 50 °C for 30 min. c MpCAPP priming is purine ribonucleotide dependent. In all, 1 µM MpCAPP was added into 1 µM mixed-sequence ssDNA substrate (oKZ53) in presence of 2.5 µM dNTPs (FAM-labelled dCTP) and 1 mM unlabelled NTPs as indicated in the figure. The reaction was incubated at 50 °C for 10 min. C – control reaction without protein, no – no NTPs, all – all NTPs, black arrow – signal of Cy5-labelled template. d DbCAPP primase activity is stimulated by addition of purine ribonucleotides. In total, 1 µM DpCAPP protein was added into 1 µM ssDNA (oKZ53) in presence of 2.5 µM dNTPs (FAM-labelled dCTP) and 1 mM unlabelled NTPs in DbPrimBuffer. The reaction was incubated at 50 °C for 10 min. Annotations identical as for panel c . nts – nucleotide length of DNA markers. Results shown are representative of three independent repeats (3a–d).

Article Snippet: MpCAPP primase assay: 20 μl reaction contained MpPrimBuffer (10 mM Bis-Tris Propane; pH 7, 0.5 mM TCEP, 10 mM MgCl 2 and 100 µM ZnCl 2 ), either 10 ng/µl circular M13 ssDNA (NEB) or 1 µM ssDNA substrate, MpCAPP proteins, unlabelled NTPs and dNTPs (NEB) and either FAM-labelled dUTP, FAM-labelled UTP, FAM-labelled dCTP or γ-phosphate Atto488-labelled GTP (NU-803-6FM, NU-821-6FM, NU-809-5FM, NU-834-488, Jena Bioscience) at combinations and concentrations indicated in the figure legends.

Techniques: Incubation, Sequencing, Activity Assay

Journal: Open Biology

Article Title: Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile

doi: 10.1098/rsob.160272

Figure Lengend Snippet: Helicase activity is stimulated by primase. Helicase activity was assayed by quantifying the displacement of a radiolabelled (γ 32 P-ATP) oligonucleotide partially annealed to the single stranded circular DNA m13mp18. Per cent displaced signal from the helicase assays in time. ( a ) Helicase activity of the CD3657 helicase with and without the CD1454 primase. ( b ) Helicase activity of the CD3657 helicase in the presence of the putative loader protein CD3654, in the presence and absence of the CD1454 primase. ( c ) Helicase activity of the CD3657 helicase in the presence of a representative Walker A mutant (T199A) of the putative loader protein CD3654, in the presence and absence of the CD1454 primase. The other mutants of CD3654 (K198R, D258Q) gave similar results (electronic supplementary material, figure S7) but have been omitted for clarity. Error bars indicate standard deviation ( n = 3).

Article Snippet: Helicase activity was assayed by monitoring (and quantifying) the displacement of the radiolabelled (γ 32 P-ATP) 105-mer oligonucleotide oVP-1 (partially) annealed to the single-stranded circular DNA m13mp18 (ssM13; Affymetrix) essentially as previously described [ ].

Techniques: Activity Assay, Mutagenesis, Standard Deviation