To better understand the dynamic regulation of microtubule structures in yeast, we studied a conditional-lethal ,B-tubulin mutation tub2-150. This mutation is unique among the hundreds of tubulin mutations isolated in Saccharomyces cerevisiae in that it appears to cause an increase in the stability of microtubules. We report here that this allele is a mutation of threonine 238 to alanine, and that tub2-150 prevents the spindle from elongating during anaphase, suggesting a nuclear microtubule defect. To identify regulators of microtubule stability and/or anaphase, yeast genes were selected that, when overexpressed, could suppress the tub2-150 temperature-sensitive phenotype. One of these genes, JSNI, encodes a protein of 125 kDa that has limited similarity to a number of proteins of unknown function. Overexpression of the JSNI gene in a TUB2 strain causes that strain to become more sensitive to benomyl, a microtubule-destabilizing drug. Of a representative group of microtubule mutants, only one other mutation, tub2-404, could be suppressed by JSNI overexpression, showing that JSNI is an allelespecific suppressor. As tub2-404 mutants are also defective for spindle elongation, this provides additional support for a role for JSNI during anaphase.
Mitotic recombination between his3 heteroalleles on heterologous chromosomes is stimulated by a DNA double chain break delivered in vivo at a site 8.6 kilobase pairs distant from one his3 allele and unlinked to the other. The induced recombination at his3 is accompanied by gap repair at the break site using the uncut homolog as a template. The DNA between the break site and his3 is not deleted in most ofthe His+ recombinants.In Saccharomyces cerevisiae a DNA double chain break is repaired by homologous recombination (1-4). It has been imagined that the ends, after some exonucleolytic degradation, invade the intact homolog, the 3'-OH ends prime synthesis that repairs the gap, and the resulting dimer is resolved, sometimes producing recombinant products (3). In this double chain break repair (DCBR) model the site of the double chain break is coincident with the site of recombination. In phage A Red recombination, a double chain end is thought to be resected on one chain to promote a long region of heteroduplex DNA (5-8). By contrast, in the major Escherichia coli recombination pathway the RecBCD enzyme apparently enters the DNA helix at the site of a double chain break, travels along the molecule, encounters a specific DNA sequence termed Chi, and stimulates recombination in the neighborhood of Chi (9, 10). Thus, in the RecBCD pathway the site ofthe exchange is arbitrarily distant from the site of the double chain break. In fungi no evidence exists for a mechanism analogous to that in the RecBCD pathway; however, there is evidence of spatially discrete but statistically correlated multiple events of recombination (11)(12)(13)(14), some of which are difficult to reconcile with the current models of recombination (11,13,14).In this communication we demonstrate that in vegetative cells of S. cerevisiae a double chain break-induced repair event is accompanied by ectopic recombination 8.6 kilobase pairs (kb) away. These coincident recombination events, involving three chromosomes, yield products that are not altered between the two sites of recombination.
MATERIALS AND METHODSStrains and Methods. Yeast strains are listed in Table 1 and their construction is described in Fig. 1. Media and biochemical methods are as described in Ray et al. (17) and in ref. 19. YEPL is YEP broth with 3 M lactate (pH 6.5). The E. coli strain was DH1 (15).Induction of HO. A single colony of the appropriate strain growing on SD-ura was suspended in YEPL broth and incubated with shaking for 8 hr. Aliquots were plated on YEP-gal, and the plates were incubated for 18 hr. Cells were washed off the plates, washed and diluted in water, and
RESULTSDouble Chain Break in cis to his3-D Stimulates Ectopic Recombination. A difference between the current view of DCBR recombination in yeast (3) and that for RecBCDcatalyzed recombination in E. coli (1, 9, 10) is that in the latter process there is no degradation of DNA between the site of double chain break and the site of initiation ofrecombination. In yeast, can recombination initiated by a doub...
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