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. 2002 Oct 1;16(19):2479–2484. doi: 10.1101/gad.232902

Figure 5.

Figure 5

2D gel analysis of rDNA ARS activity in wild-type and sir2Δ cells. Genomic DNA was prepared from congenic wild-type (E1000) and sir2Δ (E1244) cells, and replication intermediates were examined by 2D gel electrophoresis. (A) Schematic presentation of replication intermediates expected for a StuI–XbaI fragment (3.5 kb) containing the ARS element. (Bubble arc) active origins; (Y-arc) passive replication. (B) The proportion of active origins (arrowhead) increases by 60% in sir2Δ cells, but wild-type levels are restored when SIR2 is ectopically expressed in sir2Δ cells from a galactose-inducible promoter. Recombination intermediates are 20 times less abundant than replication bubbles in wild-type cells (Zou and Rothstein 1997; Ivessa et al. 2000) and are not detectable here. (C) Replication intermediates expected for an XbaI fragment (5.5 kb) centered around the RFB. X-shaped molecules correspond to converging replication forks. (D) Stalled and converged forks (arrowheads) are 43% more abundant in sir2Δ cells than in wild-type cells. (E) Quantitation by phosphor imaging of the relative amount of bubbles (lane 1) and stalled forks (lane 2) in wild-type (light gray) and sir2Δ (dark gray) cells. (F) Extrachromosomal rDNA circles (ERCs) accumulate in sir2Δ cells. Undigested genomic DNA from wild-type and sir2Δ cells was separated on a 0.7% agarose gel, transferred onto a membrane, and probed with rDNA. (Arrowheads) ERCs; (arrow) rDNA array. (G) The orc2-1 mutation reduces the accumulation of ERCs in sir2Δ cells. Genomic DNA from wild-type (E1000), sir2Δ (E1244), fob1Δ (E1410), orc2-1 (E1314), and sir2Δ orc2-1 (E1612) cells was analyzed as described in Supplementary Figure 3 (see Supplementary Material at http://www.genesdev.org), and the fraction of rDNA in circles was quantitated by phosphor imaging.