Journal: bioRxiv

Article Title: The S-phase Cyclin Clb5 Promotes rDNA Stability by Maintaining Replication Initiation Efficiency in rDNA

doi: 10.1101/2020.07.06.190892

Figure Lengend Snippet: (A) Restriction map of the rDNA repeat. Organization of the rDNA repeats is indicated as in . N and Bg indicate the sequences recognized by restriction enzymes Nhe I and Bgl II, respectively. Black and red bars represent the Southern blotting probes used for 2D and DSB analyses, respectively. (B, C) 2D agarose gel electrophoresis. Genomic DNA was digested with Nhe I. DNA was separated by size in the first dimension and by size and shape in the second dimension, and subjected to Southern blotting with the rDNA probe indicated in (A). The expected migration pattern of different replication intermediates by 2D analysis is shown in (B). 1N and 2N indicate, respectively, one- and two-unit length linear DNA molecules. Arrows in (C) indicate a double Y spot. (D, E, F) Frequency of bubble arcs (D), arrested forks (E), and double Y spots (F) determined by quantifying the signal of each intermediate relative to the total amount of replication intermediates. The levels of the different molecules in each mutant were normalized to the average of WT clones (bars indicate the range of two independent experiments). (G) Frequency of recombination intermediates at the RFB site determined by the ratio of the double Y spot signal to the bubble arc signal, which was normalized to the average of WT clones (bars indicate the range of two independent experiments). (H) Detection of arrested forks and DSBs. Genomic DNA was digested with Bgl II and separated by single-dimension agarose gel electrophoresis, followed by Southern blotting with the rDNA probe indicated in (A). Bands corresponding to arrested forks, linear fragments, DSBs, and resected DSBs are indicated. Open circle represents the terminal fragment containing the telomere-proximal rDNA repeat and its adjacent non-rDNA fragment. The lower panel shows a more exposed contrast image of the phosphorimager signal in the region marked by the dashed line. M indicates λ DNA- BstE II markers. (I) Frequency of DSBs determined as the ratio of DSB signal to arrested fork signal, which was normalized to the average of WT clones (bars indicate ranges of two independent experiments).

Article Snippet: After discarding the buffer completely, the plug was incubated in 160 μl of 1× NEBuffer 3.1 buffer containing 160 units of Bgl II (New England BIolabs) overnight at 37°C.

Techniques: Southern Blot, Agarose Gel Electrophoresis, Migration, Mutagenesis, Clone Assay