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