Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene.

Thrash, Catherine; Bankier, Alan T.; Barrell, Bart; Sternglanz, Rolf

doi:10.1073/pnas.82.13.4374

Cited by 174 publications

(110 citation statements)

References 21 publications

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“…7, we purchased a KanMX substitution of MUS81 (Research Genetics, Huntsville, AL) and generated one-step disruptions of APN1, APN2, MRE11, RAD1, and TOP1. For apn1 and top1, we used, respectively, plasmids in which the gene is disrupted by addition of a URA3 cassette (9) or carries an 849-bp deletion and a LEU2 cassette (10). For the other genes, as before (7) we used a PCR protocol to generate a complete deletion with either LEU2 (for apn2) or MET15 (for rad1 and mre11) as the substituted selectable marker.…”

Section: Methodsmentioning

confidence: 99%

Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1

Liu

Pouliot

Nash

2002

Proc. Natl. Acad. Sci. U.S.A.

160

175

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Accidental or drug-induced interruption of the breakage and reunion cycle of eukaryotic topoisomerase I (Top1) yields complexes in which the active site tyrosine of the enzyme is covalently linked to the 3 end of broken DNA. The enzyme tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes this protein-DNA link and thus functions in the repair of covalent complexes, but genetic studies in yeast show that alternative pathways of repair exist. Here, we have evaluated candidate genes for enzymes that might act in parallel to Tdp1 so as to generate free ends of DNA. Despite finding that the yeast Apn1 protein has a Tdp1-like biochemical activity, genetic inactivation of all known yeast apurinic endonucleases does not increase the sensitivity of a tdp1 mutant to direct induction of Top1 damage. In contrast, assays of growth in the presence of the Top1 poison camptothecin (CPT) indicate that the structure-specific nucleases dependent on RAD1 and MUS81 can contribute independently of TDP1 to repair, presumably by cutting off a segment of DNA along with the topoisomerase. However, cells in which all three enzymes are genetically inactivated are not as sensitive to the lethal effects of CPT as are cells defective in double-strand break repair. We show that the MRE11 gene is even more critical than the RAD52 gene for double-strand break repair of CPT lesions, and comparison of an mre11 mutant with a tdp1 rad1 mus81 triple mutant demonstrates that other enzymes complementary to Tdp1 remain to be discovered.

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Section: Methodsmentioning

confidence: 99%

Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1

Liu

Pouliot

Nash

2002

Proc. Natl. Acad. Sci. U.S.A.

160

175

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“…Mutants with moderate to high LOH at MET15 that was predominantly reciprocal (.60%) are enriched for deletions of genes involved in DNA replication and/or replication-associated chromatin assembly. Seven of the 11 mutants fall into these categories ( Figure 9): two subunits of DNA polymerases (POL32 and DPB3), two subunits of chromatin assembly factor I (MSI1 and RLF2), a replication helicase (RRM3), a subunit of an alternative replication factor C complex (ELG1), and DNA topoisomerase I (TOP1) (Thrash et al 1985;Araki et al 1991;Kaufman et al 1997;Gerik et al 1998;Ivessa et al 2002;Kanellis et al 2003). An additional member of this group is RAD6, an ubiquitin-conjugating enzyme that has both PCNA (a replication protein) and histone H2B as substrates (Robzyk et al 2000;Hoege et al 2002).…”

Section: Loh On Chromosome IIImentioning

confidence: 99%

A Genetic Screen for Increased Loss of Heterozygosity inSaccharomyces cerevisiae

et al. 2008

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Loss of heterozygosity (LOH) can be a driving force in the evolution of mitotic/somatic diploid cells, and cellular changes that increase the rate of LOH have been proposed to facilitate this process. In the yeast Saccharomyces cerevisiae, spontaneous LOH occurs by a number of mechanisms including chromosome loss and reciprocal and nonreciprocal recombination. We performed a screen in diploid yeast to identify mutants with increased rates of LOH using the collection of homozygous deletion alleles of nonessential genes. Increased LOH was quantified at three loci (MET15, SAM2, and MAT ) on three different chromosomes, and the LOH events were analyzed as to whether they were reciprocal or nonreciprocal in nature. Nonreciprocal LOH was further characterized as chromosome loss or truncation, a local mutational event (gene conversion or point mutation), or break-induced replication (BIR). The 61 mutants identified could be divided into several groups, including ones that had locusspecific effects. Mutations in genes involved in DNA replication and chromatin assembly led to LOH predominantly via reciprocal recombination. In contrast, nonreciprocal LOH events with increased chromosome loss largely resulted from mutations in genes implicated in kinetochore function, sister chromatid cohesion, or relatively late steps of DNA recombination. Mutants of genes normally involved in early steps of DNA damage repair and signaling produced nonreciprocal LOH without an increased proportion of chromosome loss. Altogether, this study defines a genetic landscape for the basis of increased LOH and the processes by which it occurs.

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“…Yeast topoisomerase I efficiently relaxes the positive and negative DNA supercoils generated, respectively, in front of and behind the molecular ensembles that track along the double helix (6 -8). Because yeast null top1 mutants are viable (9,10), the cellular roles of topoisomerase I must be fulfilled by yeast topoisomerase II, which is also efficient in the relaxation of positive and negative supercoils (Fig. 1).…”

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confidence: 99%

Failure to Relax Negative Supercoiling of DNA Is a Primary Cause of Mitotic Hyper-recombination in Topoisomerase-deficient Yeast Cells

Trigueros

Roca

2002

Journal of Biological Chemistry

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In the yeast Saccharomyces cerevisiae, DNA topoisomerases I and II can functionally substitute for each other in removing positive and negative DNA supercoils. Yeast ⌬top1 top2(ts) mutants grow slowly and present structural instability in the genome; over half of the rDNA repeats are excised in the form of extrachromosomal rings, and small circular minichromosomes strongly multimerize. Because these traits can be reverted by the extrachromosomal expression of either eukaryotic topoisomerase I or II, their origin is attributed to the persistence of unconstrained DNA supercoiling. Here, we examine whether the expression of the Escherichia coli topA gene, which encodes the bacterial topoisomerase I that removes only negative supercoils, compensates the phenotype of ⌬top1 top2(ts) yeast cells. We found that ⌬top1 top2(ts) mutants expressing E. coli topoisomerase I grow faster and do not manifest rDNA excision and minichromosome multimerization. Furthermore, the recombination frequency in repeated DNA sequences, which is increased by nearly two orders of magnitude in ⌬top1 top2(ts) mutants relative to the parental TOP؉ cells, is restored to normal levels when the bacterial topoisomerase is expressed. These results indicate that the suppression of mitotic hyper-recombination caused by eukaryotic topoisomerases I and II is effected mainly by the relaxation of negative rather than positive supercoils; they also highlight the potential of unconstrained negative supercoiling to promote homologous recombination.The budding yeast Saccharomyces cerevisiae has three structurally different DNA topoisomerases: topoisomerase I, a type IB enzyme encoded by TOP1; topoisomerase II, a type II enzyme encoded by TOP2; and topoisomerase III, a type IA enzyme encoded by TOP3 (For recent reviews see Refs. 1 and 2). Topoisomerase II is essential for cell viability because it is required to unlink intertwined pairs of replicated DNA domains at the time of mitosis (3). Another important role of topoisomerases is to prevent excessive supercoiling of DNA (4, 5). Yeast topoisomerase I efficiently relaxes the positive and negative DNA supercoils generated, respectively, in front of and behind the molecular ensembles that track along the double helix (6 -8). Because yeast null top1 mutants are viable (9, 10), the cellular roles of topoisomerase I must be fulfilled by yeast topoisomerase II, which is also efficient in the relaxation of positive and negative supercoils (Fig. 1). Conversely, the less abundant topoisomerase III does not have significant effects on the overall supercoiling of intracellular DNA (11).Genetic and biochemical studies have revealed that the DNA relaxation activity of yeast topoisomerases I and II has deep implications in genome stabilization (5, 12). Null top1 mutants have a high frequency of mitotic recombination in the rDNA cluster relative to TOPϩ cells (13). Single top2(ts) mutants, in which the weak topoisomerase II activity is abolished at 35°C, also show increased recombination in the rDNA (13) when grown at a perm...

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Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene.

Cited by 174 publications

References 21 publications

Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1

Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1

A Genetic Screen for Increased Loss of Heterozygosity inSaccharomyces cerevisiae

Failure to Relax Negative Supercoiling of DNA Is a Primary Cause of Mitotic Hyper-recombination in Topoisomerase-deficient Yeast Cells

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