We have previously shown that cells mutant for TOP3, a gene encoding a prokaryotic-like type I topoisomerase in Saccharomyces cerevisiae, display a pleiotropic phenotype including slow growth and genome instability. We identified a mutation, sgsl (slow growth suppressor), that suppresses both the growth defect and the increased genomic instability of top3 mutants. Here we report the independent isolation of the SGS1 gene in a screen for proteins that interact with Top3. DNA sequence analysis reveals that the putative Sgsl protein is highly homologous to the helicase encoded by the Escherichia coli recQ gene. These results imply that Sgsl creates a deleterious topological substrate that Top3 preferentially resolves. The interaction of the Sgsl helicase homolog and the Top3 topoisomerase is reminiscent of the recently described structure of reverse gyrase from Sulfolobus acidocaldarius, in which a type I DNA topoisomerase and a helicase-like domain are fused in a single polypeptide.Topoisomerases are ubiquitous enzymes essential for many aspects of DNA metabolism, including replication, transcriptional activation or repression, chromosome segregation, and genome stability (2,5,6,15,22,26,28,41,49). Changes in the expression of topoisomerases result in a pleiotropic phenotype in bacteria and yeast cells (33,50). In bacteria, there are four known topoisomerases. Mutations in three of these, topA, gyrAlgyrB, and parC/parE, cause changes in superhelical structure of DNA that affect the growth of the cell, transcription, transposition of transposon elements and replication as well as segregation of plasmids and daughter chromosomes (21,43,50). The fourth topoisomerase (topB) has been characterized in vitro (10,11,44) and shown to be involved in repetitive sequence stability in vivo (40).In Saccharomyces cerevisiae, topoisomerase mutations result in defects in nuclear division, transcription, recombination, and the supercoiling of plasmids. The TOP2 gene product is essential during mitosis and meiosis for the proper segregation of daughter chromosomes (12,19,20,36

Yeast cells mutant for TOP3, the gene encoding the evolutionary conserved type I-5Ј topoisomerase, display a wide range of phenotypes including altered cell cycle, hyper-recombination, abnormal gene expression, poor mating, chromosome instability and absence of sporulation. In this report, an analysis of the role of TOP3 in the meiotic process indicates that top3Δ mutants enter meiosis and complete the initial steps of recombination. However, reductional division does not occur. Deletion of the SPO11 gene, which prevents recombination between homologous chromosomes in meiosis I division, allows top3Δ mutants to form viable spores, indicating that Top3 is required to complete recombination successfully. A topoisomerase activity is involved in this process, since expression of bacterial TopA in yeast top3Δ mutants permits sporulation. The meiotic block is also partially suppressed by a deletion of SGS1, a gene encoding a helicase that interacts with Top3. We propose an essential role for Top3 in the processing of molecules generated during meiotic recombination.

Saccharomyces cerevisiu cells that are mutated at TOP3, a gene that encodes a protein homologous to bacterial type I topoisomerases, have a variety of defects, including reduced growth rate, altered gene expression, blocked sporulation, and elevated rates of mitotic recombination at several loci. The rate of ectopic recombination between two unlinked, homeologous loci, SAMI and SAM2, is sixfold higher in cells containing a top3 null mutation than in wild-type cells. Mutations in either of the two other known topoisomerase genes in S. cerevisiae, TOP] and TOP2, do not affect the rate of recombination between the S"M genes. The top3 mutation also changes the distribution of recombination events between the SAM genes, leading to the appearance of novel deletion-insertion events in which conversion tracts extend beyond the coding sequence, replacing the DNA flanking the 3' end of one SAM gene with nonhomologous DNA flanking the 3' end of the other. The effects of the top3 null mutation on recombination are dependent on the presence of an intact RADI excision repair gene, because both the rate ofSAM ectopic gene conversion and the conversion tract length were reduced in radl top3 mutant cells compared with top3 mutants. These results suggest that a R4DI-dependent function is involved in the processing of damaged DNA that results from the loss ofTop3 activity, targeting such DNA for repair by recombination.Topoisomerases are required for a variety of cellular processes, including DNA replication, transcription, recombination, and chromosome condensation and segregation (16,17). Topoisomerases from several unicellular and multicellular eukaryotes have been identified, including those of the budding yeast Saccharomyces cerevisiae (16). The genes encoding topoisomerase I (TOPJ) and topoisomerase II (TOP2) from S. cerevisiae have been cloned (5,6,14). Recently the gene encoding a putative third topoisomerase from S. cerevisiae was identified (15). This gene, TOP3, is unique among eukaryotic topoisomerase genes in that it is homologous to bacterial type I topoisomerase genes (e.g., Escherichia coli topA and topB) but not to other eukaryotic topoisomerase genes (15). The functional homology of the E. coli topA gene and S. cerevisiae TOP3 was demonstrated by the ability of the topA gene, when overexpressed, to complement the slow-growth defect of top3 mutants (15). More recently, Top3 was shown to possess a weak negative supercoil relaxing activity (6a).Topl and Top2 have been implicated in the control of mitotic recombination within the rDNA multiple tandem array but not elsewhere in the S. cerevisiae genome (4).More recently, top3 mutants, originally isolated on the basis of their increased rate of recombination between dispersed, repetitive 8 elements (15), were also found to have elevated levels of recombination within the rDNA array (4b).In event that gives rise to genome rearrangements is also observed in top3 mutants. Altered recombination in the top3 mutant is at least partially dependent on the presence of a wild-ty...

We have previously shown that cells mutant for TOP3, a gene encoding a prokaryotic-like type I topoisomerase in Saccharomyces cerevisiae, display a pleiotropic phenotype including slow growth and genome instability. We identified a mutation, sgs1 (slow growth suppressor), that suppresses both the growth defect and the increased genomic instability of top3 mutants. Here we report the independent isolation of the SGS1 gene in a screen for proteins that interact with Top3. DNA sequence analysis reveals that the putative Sgs1 protein is highly homologous to the helicase encoded by the Escherichia coli recQ gene. These results imply that Sgs1 creates a deleterious topological substrate that Top3 preferentially resolves. The interaction of the Sgs1 helicase homolog and the Top3 topoisomerase is reminiscent of the recently described structure of reverse gyrase from Sulfolobus acidocaldarius, in which a type I DNA topoisomerase and a helicase-like domain are fused in a single polypeptide.

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