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: Nature Communications

Article Title: DNA polymerase ε relies on a unique domain for efficient replisome assembly and strand synthesis

doi: 10.1038/s41467-020-16095-x

Figure Lengend Snippet: a Doxycycline addition acutely reduces Dpb11 levels. Western blots verified Dpb11 loss in G1- arrested cells and then cells released into S phase when treated with doxycycline (+Dox), which turned off Tet promoter-driven Dpb11 expression . b Dpb11 depletion delays origin firing but does not generate asymmetric replication intermediates. Representative 2D gel pictures are shown. Cells were treated as in panel a; 2D gel analyses were performed and data are presented as in Fig. . c Quantification of the bubble-shaped replication intermediates shown in panel b. d Reconstituted DNA replication reactions were performed on pARS1 (4.8 kb) in the presence of increasing concentrations of Pol ε WT (lanes 1–4) or Pol ε REL (lanes 5–8) as indicated. Replication products were analyzed by alkaline agarose gel-electrophoresis and the total incorporation of 32 P-dATP is plotted (bottom). Lead: leading strands, lag: lagging strands. e Primer extension assay with purified Pol ε WT (lanes 1–7) or Pol ε REL (lanes 8–14). Single-stranded M13mp18 DNA (7.2 kb) was primed with a radio-labeled oligo, incubated with RFC/PCNA, and reactions initiated by addition of Pol ε and dNTPs. Products were analyzed by alkaline agarose gel-electrophoresis and autoradiography. FL: full length. f Time-course analysis of reconstituted DNA replication reactions in the presence of 15 nM Pol ε WT (lanes 1–5) or Pol ε REL (lanes 6–10). Replication products were analyzed and quantified as in c. For panel a and b, similar results were obtained using at least two independent strains per genotype. For panel c, d similar results were obtained from two independent experiments. Source data are provided as a Source data file.

Article Snippet: The reactions contained 1 nM circular DNA template (M13mp18 ssDNA, NEB) annealed to a radio-labeled oligo ( 32 P-5ʹ-CCCAGTCACGACGTTGTAAAACG), 60 nM PCNA, 10 nM RFC, 400 nM RPA, 5 mM ATP, 2.5 mM DTT, 0.1 mg ml −1 BSA and 100 nM of either Pol ε WT or Pol ε REL .

Techniques: Western Blot, Expressing, Two-Dimensional Gel Electrophoresis, Agarose Gel Electrophoresis, Primer Extension Assay, Purification, Labeling, Incubation, Autoradiography