Genome rearrangement in top3 mutants of Saccharomyces cerevisiae requires a functional RAD1 excision repair gene.

Abstract

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...

Influence of DNA Sequence Identity on Efficiency of Targeted Gene Replacement

“…The sam1-⌬BglII frameshift and sam1:: LEU2 deletion/disruption mutations were constructed and used as previously described (3)(4)(5). The sam2-⌬SalI::HIS3 and sam2-⌬SalI::CYH2::URA3 gene disruption constructions described above were transplaced into the yeast genome by single-step gene disruption (33).…”

“…AdoMet prototrophs can arise as the result of several different events. In addition to recombination between the SAM substrate fragment on the plasmid and the SAM2 locus, gene conversion between the SAM1 and SAM2 loci, as well as spontaneous reversion of the mutations at either of the SAM loci, can result in AdoMet prototrophy (3)(4)(5). The opposite orientations of the SAM1 and SAM2 genes relative to their centromeres preclude the isolation of reciprocal recombinants (5).…”

The Essential Helicase Gene RAD3 Suppresses Short-Sequence Recombination in Saccharomyces cerevisiae

“…Determining the nature of the event that resulted in AdoMet prototrophy requires digesting the DNA with enzymes that give diagnostic fragments if the insertions that inactivate the SAM genes are replaced with wild-type information (5,6). Coconversion events are documented by digesting DNA from the recombinants with PvuII before blotting and hybridization ( Fig.…”

A Delicate Balance Between Repair and Replication Factors Regulates Recombination Between Divergent DNA Sequences inSaccharomyces cerevisiae

“…Recently Sgs1 and Top3-Rmi1 were shown to have interdependent and independent functions in meiosis (Kaur et al 2015;Tang et al 2015). However an antirecombination role for the Top3-Rmi1 complex has not been clearly established, though previous studies showed that top3D mutants increased recombination between divergent DNA sequences (Wallis et al 1989;Bailis et al 1992).…”