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The hypothesis that DNA topoisomerase II facilitates the separation of replicated sister chromatids was tested by examining the consequences of chromosome segregation in the absence of topoisomerase H activity. We observed a substantial elevation in the rate of nondisjunction in top2/top2 cells incubated at the restrictive temperature for one generation time. In contrast, only a minor increase in the amount of chromosome breakage was observed by either physical or genetic assays. These results suggest that aneuploidy is a major cause of the nonviability observed when top2 cells undergo mitosis at the restrictive temperature. In related experiments, we determined that topoisomerase II must act specifically during mitosis. This latter observation is consistent with the hypothesis that the mitotic spindle is necessary to allow topoisomerase II to complete the untangling of sister chromatids.DNA topoisomerase II plays a critical but as yet incompletely defined role in chromosome metabolism in eucaryotes. Two general experimental approaches support the conclusion that topoisomerase II is important in chromosome segregation. First, in vitro studies show that topoisomerase II has the strand-passing abilities expected of an enzyme that can untangle sister DNA molecules that are topologically linked following DNA replication. Second, temperature shift experiments in both Saccharomyces cerevisiae and Schizosaccharomyces pombe show that topoisomerase II is specifically required at the time that chromosomes are segregating from one another.Extensive in vitro studies of topoisomerase II demonstrate that the enzyme catalyzes the interconversion between various topological forms of circular DNA molecules (for recent reviews, see references 5, 9, and 39). Although type I topoisomerases also catalyze topoisomerization, their mechanism involves a single-strand nick rather than a doublestrand break (26). In contrast, type II topoisomerases act by making a double-strand break, passing another double strand through the break, and then resealing it (2, 21). Thus, in addition to catalyzing the increase or decrease in linking number of a DNA molecule, type II topoisomerases can also catalyze catenation and decatenation of circular DNA molecules, and they can tie and untie knots in them (1,14,15,(19)(20)(21). The in vitro properties of topoisomerase II are therefore consistent with a role in untangling intertwined DNA molecules in vivo.Experimental evidence that topoisomerase II is important for chromosome transactions in vivo derives from studies of circular DNA molecules and temperature-sensitive mutants. A variety of circular DNA molecules appear to be intertwined following DNA replication in the absence of topoisomerase II activity (6, 33, 34), although the degree of intertwining may be influenced by the DNA sequence at the termination point of DNA replication (40). tive top2 mutants in S. cerevisiae or S. pombe are shifted to the restrictive temperature, they rapidly lose viability only if they are passing through mitosis (13, 37)....

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In vitro studies suggest that the Barren protein may function as an activator of DNA topoisomerase II and/or as a component of theXenopus condensin complex. To better understand the role of Barren in vivo, we generated conditional alleles of the structural gene for Barren (BRN1) in Saccharomyces cerevisiae. We show that Barren is an essential protein required for chromosome condensation in vivo and that it is likely to function as an intrinsic component of the yeast condensation machinery. Consistent with this view, we show that Barren performs an essential function during a period of the cell cycle when chromosome condensation is established and maintained. In contrast, Barren does not serve as an essential activator of DNA topoisomerase II in vivo. Finally,brn1 mutants display additional phenotypes such as stretched chromosomes, aberrant anaphase spindles, and the accumulation of cells with >2C DNA content, suggesting that Barren function influences multiple aspects of chromosome transmission and dynamics.

DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30 -104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30 -104 mutation impairs MSH2/ MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.In both prokaryotes and eukaryotes, defects in DNA mismatch repair cause elevated spontaneous mutation rates and increased instability of simple repeat DNA sequences. Mutations in any of the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. Cell lines from these cancers are defective in DNA mismatch repair and display increased levels of spontaneous mutations and frequent alterations of microsatellite repeat sequences (1, 2).Epistasis analyses in yeast have suggested that MSH2 protein functions in conjunction with MSH3 or MSH6 protein in mismatch recognition. Genetic and biochemical studies in both yeast and humans have further indicated that the MSH2-MSH3 and MSH2-MSH6 complexes differ in substrate specificities. In yeast, mutations in MSH3 cause an increase in instability of microsatellite tracts but have little effect on single-base mispairs, whereas mutations in MSH6 have a more prominent effect on the incidence of single-base mispairs than on microsatellite tract instability (3-5). From these and other genetic observations, it has been inferred that MSH2-MSH3 complex is more proficient in the removal of insertion-deletion mismatches of two or more nucleotides (4), whereas MSH2-MSH6 is better at removing single nucleotide mismatches (4, 5). Human cell lines defective in the MSH6 component of the MSH2-MSH6 heterodimer hMutS␣ exhibit a selective loss in the repair of base-base and single-nucleotide insertion-deletion mismatches; the repair of two-, three-, and four-nucleotide insertion-deletion mismatches is reduced 2-4-fold in these cell lines (6, 7). Consistent with genetic observations, hMutS␣ binds a G/T mismatch or a one nucleotide insertion-deletion mismatch with high efficiency (6). By contrast, the yeast MSH2-MSH3 heterodimer exhibits little affinity for a G/T mismatch but binds insertion-deletion mismatches with high specificity (8). The manner by which PMS1 and MLH1 function in mismatch...