Replication Pausing and Imprinting Figure 5. Southern analysis of mat1 replication intermediates. (A) Line drawing of the fragments generated for different strains for denaturing polyacrylamide gel electrophoresis (PAGE) analysis of replicating mat1M DNA shown in panels B and C. The positions of the restriction enzyme sites and the mapped priming site, and the lengths of the fragments are given. The site of the imprint is indicated with a circle. (B) Denaturing PAGE analysis of the mat1M lagging strand. Replicating DNA from log-phase cells was isolated as for 2D-gel analysis, digested with restriction enzymes given in panel A, then was subjected to denaturing PAGE analysis. After blotting, the membrane was probed with a strand- specific probe that hybridizes to the nascent lagging strand and the parental upper strand. Denatured and labelled DNA ladder is given to the left with size markers. The strains that exhibit imprinting (WT and SS25) produced two strong bands that correspond to the centromere-distal and proximal DSB products created by hydrolysis of the imprint (1d and 1p, respectively). Specific priming products at the site of the imprint (2) migrate to the same position as the distal DSB product observed for the WT and SS25 strains (1d). See panel D. Two fainter putative priming products that are shared between WT and SS25 are marked by arrowheads. (C) Denaturing PAGE analysis of the mat1M leading strand. The nascent leading strand and the parental lower strand were detected by the strand-specific probe. The co-migrating signals from the leading strand intermediates stalled at the imprint in WT and SS25 strains (3) and at MPS1 in the strain SS13 (4) are indicated. See panel D. Denatured and labelled DNA ladder is given to the left with size markers. (D) Line drawings of the replication intermediates observed in panel B and C are given. Fragments that produce bands in PAGE PLoS Genetics | www.plosgenetics.org 8 March 2011 | Volume 7 | Issue 3 | e1001328