Figure 2.

Neither catenated plasmids nor broken chromosomes are apparent in brn1 strains at the restrictive temperature. (A) Plasmid catenation in top2 and brn1 mutants. Strains CH2523 (BRN1), CH2524 (brn1-9), CH2525 (brn1-16), CH335 (TOP2), and CH325 (top2-4), each containing a 14-kb CEN3 ARS1 plasmid (pDK243), were synchronized in G1 with α-factor, shifted to the restrictive temperature, and then released into prewarmed medium containing nocodazole (37°C) to rearrest cells in mitosis (M). Isolated plasmid DNA was separated by agarose gel electrophoresis and visualized by Southern blotting with the use of a pBR322-based probe. S, L, and R denote the supercoiled, linear, and relaxed forms, respectively. Catenated circular minichromosomes run as a high-molecular-weight smear of topoisomers (see top2-4, lane M). The unlabeled M-specific band between L and R has been ascribed to two closed circular intertwined monomers (C forms), as described by Koshland and Hartwell (1987). (B) Assay for broken chromosomes in brn1 and top2 mutants. Strains CH2523 (BRN1 TOP2), CH2524 (brn1-9),CH2525 (brn1-16), and CH325 (top2-4) were grown exponentially at 25°C, the cultures were divided, and half of each culture was incubated at 25°C and half at 37°C for 5 h. Chromosomes were separated on a CHEF gel, and chromosome V was visualized by hybridization with a URA3 probe. The smear of hybridization that lies between the well and the chromosome V band is not reproducible. Characteristic fragments of chromosome V appear only in strain CH325 (top2-4) at 37°C. WT, wild type. STD, chromosomal molecular weight standard.