Replication Pausing and Imprinting Figure 2. Identification of two regions required for imprinting and pausing within the mat1M cassette. (A) Line drawing on the left, a simple representation of the intact mat1M cassette flanked with the homology domains is shown. The site of the imprint is indicated by a vertical arrow. The nucleotide length of the region is given. The positions of the two encoded transcripts, Mi and Mc, are shown by horizontal arrows. The full- length wild-type DNA is represented by an intact line, whereas deletions are shown as gapped lines and the nucleotide positions of the start and the end of each deletion are given below the lines. The position of the specific lagging-strand priming site described in 12] is marked by a vertical line. The text box on right shows the names of the strains used, followed by quantification of the double-strand break (DSB) products (including standard error) and of the replication pause signal observed on 2D gels. The results are given as percentages of the wild-type values (100%). For DSB products, the values lower or equal to the negative control threshold are indicated as ‘‘below detection’’ (B.D.), whilst the absence of a pause signal is indicated by a dash. (B) Southern analysis of the strains shown in panel A. Genomic DNA was digested with HindIII and probed with the mat1P HindIII fragment. During DNA isolation, the imprint is hydrolyzed to a DSB and the resulting break products migrate as two bands. The strain names are given on top, PLoS Genetics | www.plosgenetics.org 4 March 2011 | Volume 7 | Issue 3 | e1001328