Deletion of FOB1 only partially suppresses the generation of rDNA circles in rrm3 cells. (A) Undigested DNA was separated using 2D gels. The DNA was transferred to a membrane and hybridized simultaneously with two rDNA probes. One probe is indicated in Figure 1A; the other is a fragment of 35S rDNA. Asterisks indicate the position of linear chromosomal rDNA, and arrows indicate the presence of rDNA supercoiled monomer circles. Additional rDNA circle species form an arc above the chromosomal DNA. (B) Quantitation of a representative experiment. The number above each column is the amount of hybridization in circles divided by the total rDNA hybridization.

Deletion of FOB1 only partially suppresses the rrm3 extended cell cycle. (A) G1-phase (α-factor-arrested) wild-type, rrm3, fob1, and rrm3 fob1 cells were released and allowed to progress through the cell cycle at 23°C. Aliquots were removed at indicated times in minutes and analyzed for DNA content by FACS. (B) Deletion of FOB1 does not rescue the rrm3 mrc1 synthetic lethality. Tetrad dissection of diploids homozygous for rrm3 and heterozygous for mrc1 and fob1. The four spores from a given tetrad are in a vertical line; dissected spores were grown on complete medium for 3 d at 30°C, and genotyped by replica plating. Assuming 2:2 segregation of the genetic markers allows one to identify rrm3 mrc1 double (circled) and rrm3 mrc1 fob1 triple mutants (boxed). Wild-type, rrm3, rrm3 mrc1, and rrm3 mrc1 fob1 spore products carrying pIA20 were streaked onto FOA and assessed for growth after 3 d at 30°C. Lack of growth on FOA indicates that the strain is inviable in the absence of pIA20.

Fob1p is required for rrm3-dependent pausing at the RFB but not at other rDNA sites. (A) Schematics of HaeII–SnaBI-digested rDNA. The first panel indicates replication forks initiated in upstream repeats. The middle pane indicates replication forks in repeats with an active origin and an active RFB. The right panel ndicates forks initiated in downstream repeats in an fob1 strain. These forks are only seen when there is no active RFB, as in fob1 cells. Vertical lines and lowercase letters indicate where replication forks pause in an rrm3 strain. (B) The 2D gel schematics show the pattern of replication intermediates in the indicated strains. Intermediates in gray are from forks initiated within the repeat, and intermediates in black are rightward-moving forks. When Fob1p is deleted, leftward-moving forks are also shown in black. Lowercase letters indicate the specific pause sites illustrated in A. (C) 2D gels of HaeII–SnaBI-digested rDNA. (Panel 1) Wild type. (Panel 2) rrm3. (Panel 3) fob1. (Panel 4)rrm3 fob1. The arrows in panels 2 and 4 indicate the expected position of the rightward-moving replication forks pausing at the RFB. The asterisks indicate the location of leftward-moving forks arrested at the RFB. (D–F) The same as A, B, and C except XbaI-digested DNA was examined. Pauses d-l and e-l are at the same sites as pauses d and e but are generated by leftward-moving forks moving through these sites.

rDNA replication in Saccharomyces cerevisiae. (A) Schematic of a single repeat of yeast rDNA. Shown are recognition sites for the restriction enzymes used [(Bg) BglII; (Xb) XbaI; (Ha) HaeII; (Sn) SnaBI] and the position of the probe used for all 2D gels. (B) Schematic of yeast rDNA replication (Brewer and Fangman 1988; Linskens and Huberman 1988). For simplicity the 5S rDNA gene is not shown. The large arrows indicate 35S rRNA genes, the rectangles indicate the RFB, and the circles represent the ARSs. (C) Schematics of BglII-digested rDNA. The left panel indicates the replication intermediates from forks initiated in upstream repeats. Vertical lines and lowercase letters indicate sites of replication fork pausing in rrm3 cells. The right panel shows a repeat with an active origin of DNA replication. Intermediates in gray are from leftward-moving forks and intermediates in black are from rightward-moving forks. (D) The 2D gel schematics show the pattern of replication intermediates in the indicated strains. The simple Y replication arc is a composite of rightward-moving forks from repeats without an active origin (black) and forks from repeats with an active origin (gray). Forks arrested (RFB) and converged (labeled X) at the RFB are indicated. (E) Sir2p does not contribute to rrm3-dependent pauses. 2D gels of BglII-digested rDNA from the indicated strains. (Panel 1) Wild type. (Panel 2) rrm3. (Panel 3) sir2. (Panel 4) rrm3 sir2. For these and subsequent 2D replication gels, digested DNA was Southern blot transferred and hybridized with the probe indicated in A.