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PubMed1988

Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22.

Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.

Base Sequence↗
PubMed1991

Proteolysis patterns of epitopically labeled yeast DNA topoisomerase II suggest an allosteric transition in the enzyme induced by ATP binding.

A cloned yeast TOP2 gene was modified to produce yeast DNA topoisomerase II (EC 5.99.1.3) epitopically labeled at its amino or carboxyl terminus. Limited digestion with SV8 endoprotease shows three distinct protease-sensitive sites in each polypeptide of the dimeric enzyme. These sites were mapped by immunostaining of the end-labeled proteolytic fragments resolved by SDS/polyacrylamide gel electrophoresis; two of the mapped locations were confirmed by sequencing the amino ends of two unlabeled peptic fragments. Proteolytic cleavage by SV8 endoprotease at a pair of sites corresponding to the carboxyl sides of Glu-411 and Glu-680 is modulated by the binding of the nonhydrolyzable ATP analogs adenosine 5'-[beta, gamma-imido]triphosphate (5'-adenylyl imidodiphosphate) and adenosine 5'-[gamma-thio]triphosphate: in their absence cleavage occurs predominantly at Glu-411; in the presence of either analog, cleavage occurs predominantly at Glu-680. These results are interpreted in terms of allosteric interdomainal movements in the type II DNA topoisomerase following the binding of ATP.

Adenosine Triphosphate↗
PubMed2004

A mutation in human topoisomerase II alpha whose expression is lethal in DNA repair-deficient yeast cells.

Type II DNA topoisomerases are ATP-dependent enzymes that catalyze alterations in DNA topology. These enzymes are important targets of a variety of anti-bacterial and anti-cancer agents. We identified a mutation in human topoisomerase II alpha, changing aspartic acid 48 to asparagine, that has the unique property of failing to transform yeast cells deficient in recombinational repair. In repair-proficient yeast strains, the Asp-48 --> Asn mutant can be expressed and complements a temperature-sensitive top2 mutation. Purified Asp-48 --> Asn Top2alpha has relaxation and decatenation activity similar to the wild type enzyme, but the purified protein exhibits several biochemical alterations compared with the wild type enzyme. The mutant enzyme binds both covalently closed and linear DNA with greater avidity than the wild type enzyme. hTop2alpha(Asp-48 --> Asn) also exhibited elevated levels of drug-independent cleavage compared with the wild type enzyme. The enzyme did not show altered sensitivity to bisdioxopiperazines nor did it form stable closed clamps in the absence of ATP, although the enzyme did form elevated levels of closed clamps in the presence of a non-hydrolyzable ATP analog compared with the wild type enzyme. We suggest that the lethality exhibited by the mutant is likely because of its enhanced drug-independent cleavage, and we propose that alterations in the ATP binding domain of the enzyme are capable of altering the interactions of the enzyme with DNA. This mutant enzyme also serves as a new model for understanding the action of drugs targeting topoisomerase II.

Adenosine Triphosphate↗
PubMed1993

Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast.

Fluorescence in situ hybridization (FISH) shows that fission yeast centromeres and telomeres make up specific spatial arrangements in the nucleus. Their positioning and clustering are cell cycle regulated. In G2, centromeres cluster adjacent to the spindle pole body (SPB), while in mitosis, their association with each other and with the SPB is disrupted. Similarly, telomeres cluster at the nuclear periphery in G2 and their associations are disrupted in mitosis. Mitotic centromeres interact with the spindle. They remain undivided until the spindle reaches a critical length, then separate and move towards the poles. This demonstrated, for the first time, that anaphase A occurs in fission yeast. The mode of anaphase A and B is similar to that of higher eukaryotes. In nda3 and cut7 mutants defective in tubulin of a kinesin-related motor, cells are blocked in early stages of mitosis due to the absence of the spindle, and centromeres dissociate but remain close to the SPB, whereas in a metaphase-arrested nuc2 mutant, they reside at the middle of the spindle. FISH is therefore a powerful tool for analyzing mitotic chromosome movement and disjunction using various mutants. Surprisingly, in top2 defective in DNA topoisomerase II, while most chromatid DNAs remain undivided, sister centromeres are separated. Significance of this finding is discussed. In contrast, most chromatid DNAs are separated but telomeric DNAs are not in cut1 mutant. In cut1, the dependence of SPB duplication on the completion of mitosis is abolished. In crm1 mutant cells defective in higher-order chromosome organization, the interphase arrangements of centromeres and telomeres are disrupted.

Cell Cycle↗
PubMed1994

Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidization by uncoupling chromosome dynamics from other cell cycle events.

ICRF-193, a novel noncleavable, complex-stabilizing type topoisomerase (topo) II inhibitor, has been shown to target topo II in mammalian cells (Ishida, R., T. Miki, T. Narita, R. Yui, S. Sato, K. R. Utsumi, K. Tanabe, and T. Andoh. 1991. Cancer Res. 51:4909-4916). With the aim of elucidating the roles of topo II in mammalian cells, we examined the effects of ICRF-193 on the transition through the S phase, when the genome is replicated, and through the M phase, when the replicated genome is condensed and segregated. Replication of the genome did not appear to be affected by the drug because the scheduled synthesis of DNA and activation of cdc2 kinase followed by increase in mitotic index occurred normally, while VP-16, a cleavable, complex-stabilizing type topo II inhibitor, inhibited all these processes. In the M phase, however, late stages of chromosome condensation and segregation were clearly blocked by ICRF-193. Inhibition at the stage of compaction of 300-nm diameter chromatin fibers to 600-nm diameter chromatids was demonstrated using the drug during premature chromosome condensation (PCC) induced in tsBN2 baby hamster kidney cells in early S and G2 phases. In spite of interference with M phase chromosome dynamics, other mitotic events such as activation of cdc2 kinase, spindle apparatus reorganization and disassembly and reassembly of nuclear envelopes occurred, and the cells traversed an unusual M phase termed "absence of chromosome segregation" (ACS)-M phase. Cells then continued through further cell cycle rounds, becoming polyploid and losing viability. This effect of ICRF-193 on the cell cycle was shown to parallel that of inactivation of topo II on the cell cycle of the ts top2 mutant yeast. The results strongly suggest that the essential roles of topo II are confined to the M phase, when the enzyme decatenates intertwined replicated chromosomes. In other phases of the cycle, including the S phase, topo II may thus play a complementary role with topo I in controlling the torsional strain accumulated in various genetic processes.

Animals↗
PubMed2005

Proteomic and genomic characterization of chromatin complexes at a boundary.

We have dissected specialized assemblies on the Saccharomyces cerevisiae genome that help define and preserve the boundaries that separate silent and active chromatin. These assemblies contain characteristic stretches of DNA that flank particular regions of silent chromatin, as well as five distinctively modified histones and a set of protein complexes. The complexes consist of at least 15 chromatin-associated proteins, including DNA pol epsilon, the Isw2-Itc1 and Top2 chromatin remodeling proteins, the Sas3-Spt16 chromatin modifying complex, and Yta7, a bromodomain-containing AAA ATPase. We show that these complexes are important for the faithful maintenance of an established boundary, as disruption of the complexes results in specific, anomalous alterations of the silent and active epigenetic states.

Chromatin↗
PubMed1999

Rad18 is required for DNA repair and checkpoint responses in fission yeast.

To survive damage to the genome, cells must respond by activating both DNA repair and checkpoint responses. Using genetic screens in the fission yeast Schizosaccharomyces pombe, we recently isolated new genes required for DNA damage checkpoint control. We show here that one of these strains defines a new allele of the previously described rad18 gene, rad18-74. rad18 is an essential gene, even in the absence of extrinsic DNA damage. It encodes a conserved protein related to the structural maintenance of chromosomes proteins. Point mutations in rad18 lead to defective DNA repair pathways responding to both UV-induced lesions and, as we show here, double-stranded breaks. Furthermore, rad18p is required to maintain cell cycle arrest in the presence of DNA damage, and failure of this leads to highly aberrant mitoses. A gene encoding a BRCT-containing protein, brc1, was isolated as an allele-specific high-copy suppressor of rad18-74. brc1 is required for mitotic fidelity and for cellular viability in strains with rad18 mutations but is not essential for DNA damage responses. Mutations in rad18 and brc1 are synthetically lethal with a topoisomerase II mutant (top2-191), indicating that these proteins play a role in chromatin organization. These studies show a role for chromatin organization in the maintenance or activation of responses to DNA damage.

Adenosine Triphosphate↗
PubMed1995

Isolation of mutants of Saccharomyces cerevisiae requiring DNA topoisomerase I.

Despite evidence that DNA topoisomerase I is required to relieve torsional stress during DNA replication and transcription, yeast strains with a top1 null mutation are viable and display no gross defects in DNA or RNA synthesis, possibly because other proteins provide overlapping functions. We isolated mutants whose inviablility or growth defect is relieved when TOP1 is expressed [trf mutants (topoisomerase one-requiring function)]. The TRF genes define at least four complementation groups. TRF3 is allelic to TOP2. TRF1 is allelic to HPR1, previously shown to be homologous to TOP1 over two short regions. TRF4 encodes a novel 584-amino acid protein with homology to the N-terminus of Saccharomyces cerevisiae topo I. Like top1 mutants, trf4 mutants have elevated rDNA recombination and fail to shut off RNA polymerase II transcription in stationary phase. trf4 null mutants are cs for viability, display reduced expression of GAL1 and Cell Cycle Box UAS::LacZ fusions, and are inviable in combination with trfI null mutants, indicating that both proteins may share a common function with DNA topoisomerase I. The existence of multiple TRF complementation groups suggests that not all biological functions of topo I can be carried out by topo II.

Alleles↗
PubMed1992

Characterization of a topoisomerase II gene rearrangement in a human small-cell lung cancer cell line.

BACKGROUND: Small-cell lung cancer (SCLC) is a highly chemosensitive tumor, but the recurrent disease that is common after initial response is often unresponsive to further chemotherapy. Although the mechanisms of drug resistance in SCLC have not been established, studies suggest that alterations of the nuclear enzyme DNA topoisomerase II may reduce the sensitivity of the cell to drug action. This enzyme is recognized as a primary target for cytotoxic activity of important antitumor agents. PURPOSE: In this study, we attempted to determine if altered forms of DNA topoisomerase II are responsible for reduced drug sensitivity. METHODS: We characterized a rearrangement of the topoisomerase II p170 gene (also known as TOP2) in a relatively chemoresistant SCLC cell line, NCI-H69, and compared topoisomerase II expression and activity in this line with those in the chemosensitive NCI-H187 cell line. Fragments of complementary DNA from the topoisomerase II gene were generated by polymerase chain reaction. Immunodetection was accomplished by using the monoclonal antibody 7E6 against the human topoisomerase II p170 isoform. Using DNA probes corresponding to different complementary DNA regions, we showed that the rearrangement was localized at the 3' terminus of one allele of the topoisomerase II gene. RESULTS: In addition to the normal 6.2-kilobase (kb) topoisomerase II messenger RNA (mRNA), the NCI-H69 line expressed a 7.4-kb topoisomerase II transcript, presumably encoded by the rearranged allele. Moreover, this transcript, although longer than the normal mRNA, lacked a substantial portion of the 3'-terminal p170 gene coding sequence. Topoisomerase II activity in nuclear extracts, as determined by the P4 phage DNA-unknotting assay, was more easily detected and measured at lower NaCl concentrations in NCI-H69 than in NCI-H187 cells. CONCLUSION: These results are consistent with the hypothesis that the chemoresistant NCI-H69 cell line may express, in addition to the normal enzyme, an altered topoisomerase II enzyme possibly encoded by the 7.4-kb mRNA, which in turn may be transcribed from the rearranged gene allele. IMPLICATION: These observations emphasize the role of topoisomerase II in determining drug sensitivity and suggest that such gene rearrangements may contribute to resistance of SCLC cells to topoisomerase II inhibitors.

Alleles↗
PubMed2001

Characterisation of the gene encoding type II DNA topoisomerase from Leishmania donovani: a key molecular target in antileishmanial therapy.

The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflagellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. DNA sequence analysis revealed an ORF of 3711 bp encoding a putative protein of 1236 amino acids with no introns. The deduced amino acid sequence of LdTOP2 showed strong homologies to TOP2 sequences from other kinetoplastids, namely Crithidia and Trypanosoma spp. with estimated identities of 86 and 68%, respectively. LdTOP2 shares a much lower identity of 32% with its human homologue. LdTOP2 is located as a single copy on a chromosome in the 0.7 Mb region in the L.donovani genome and is expressed as a 5 kb transcript. 5'-Mapping studies indicate that the LdTOP2 gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon occurring at -639 from the predicted initiation site. Antiserum raised in rabbit against glutathione S-transferase fusion protein containing the major catalytic portion of the recombinant L.donovani topoisomerase II protein could detect a band on western blots at approximately 132 kDa, the expected size of the entire protein. Use of the same antiserum for immunolocalisation analysis led to the identification of nuclear, as well as kinetoplast, antigens for L.donovani topoisomerase II. The in vitro biochemical properties of the full-length recombinant LdTOP2 when overexpressed in E.coli were similar to the Mg(II) and ATP-dependent activity found in cell extracts of L.donovani.

5' Untranslated Regions↗
PubMed2004

The alpha4 residues of human DNA topoisomerase IIalpha function in enzymatic activity and anticancer drug sensitivity.

We introduced a series of Pro substitutions within and near the alpha4 helix, a part of the breakage/rejoining region, in human DNA topoisomerase IIalpha, and analyzed if this region is involved in determination of anti-cancer drug sensitivity in a temperature- sensitive yeast strain (top2-4 allele). Among the 19 mutants generated, H759P and N770P showed resistance to etoposide and doxorubicin at the non-permissive temperature, where cell growth depends on activity of the human enzyme. For these residues, mutants with an Ala substitution were further created, in which H759A also showed resistance to etoposide. H759P, H759A and N770P were expressed, purified and subjected to in vitro measurement of drug sensitivity. They generated lower amounts of the etoposide-induced cleavable complexes, and were also found to have lower decatenation activity than the wild-type. In the crystal structure, the yeast equivalent of His759 is found in the vicinity of the Arg713, a putative anchoring residue of the 3'-side of cleaved DNA strands. These results suggest that His759 and the other alpha4 helix residues are involved in the enzymatic activity and drug sensitivity of human DNA topoisomerase IIalpha, via interaction with cleaved DNA.

Amino Acid Sequence↗
PubMed2006

Distribution of gyrase and topoisomerase IV on bacterial nucleoid: implications for nucleoid organization.

We explored the existence of nucleoid DNA loops in Escherichia coli by studying the distribution of bacterial type II topoisomerases (Topo IIs). Norfloxacin-induced high molecular weight (HMW) DNA fragmentation of nucleoid, an event reminiscent of the excision of eukaryotic chromosomal DNA loops mediated by topoisomerase II (TOP2). The size of the HMW DNA fragments induced by norfloxacin was affected by transcription, translation and growth phases of bacteria. The involvement of bacterial Topo IIs in the generation of these HMW DNA fragments is supported by the following observations: (i) the excised loop-sized DNA fragments were covalently linked to proteins; (ii) the norfloxacin-induced excision of DNA loops was highly reversible; (iii) coumermycin A1 antagonized the excision of DNA loops induced by norfloxacin; (iv) this antagonistic effect was reduced in either gyrase or topo IV mutants conferring coumarin resistance and (v) norfloxacin-induced reversible, gyrase-mediated DNA cleavage in vitro. Importantly, studies on coumarin- and/or quinolone-resistant mutant strains showed that DNA gyrase, rather than topoisomerase IV, plays the major role in the generation of loop-sized HMW DNA fragments. In sum, our study suggests a potential role of Topo IIs in the arrangement of DNA supercoiling loop domains in prokaryotic cells.

Aminocoumarins↗
PubMed2006

SUMO modifications control assembly of synaptonemal complex and polycomplex in meiosis of Saccharomyces cerevisiae.

The synaptonemal complex (SC) is a proteinaceous complex that apparently mediates synapsis between homologous chromosomes during meiotic prophase. In Saccharomyces cerevisiae, the Zip1 protein is the integral component of the SC. In the absence of a DNA double-strand break or the SC initiation protein Zip3, Zip1 proteins aggregate to form a polycomplex (PC). In addition, Zip1 is also responsible for DSB-independent nonhomologous centromere coupling at early meiotic prophase. We report here that Zip3 is a SUMO (small ubiquitin-related modifier) E3 ligase and that Zip1 is a binding protein for SUMO-conjugated products. Our results also suggest that at early meiotic prophase, Zip1 interacts with Zip3-independent Smt3 conjugates (e.g., Top2) to promote nonhomologous centromere coupling. At and after mid-prophase, the Zip1 protein begins to associate with Zip3-dependent Smt3 conjugates (e.g., Red1) along meiotic chromosomes in the wild-type cell to form SCs and with Smt3 polymeric chains in the zip3 mutant to form PCs.

Amino Acid Sequence↗
PubMed2005

Large-scale analysis of genes that alter sensitivity to the anticancer drug tirapazamine in Saccharomyces cerevisiae.

Tirapazamine (TPZ) is an anticancer drug that targets topoisomerase II. TPZ is preferentially active under hypoxic conditions. The drug itself is not harmful to cells; rather, it is reduced to a toxic radical species by an NADPH cytochrome P450 oxidoreductase. Under aerobic conditions, the toxic compound reacts with oxygen to revert back to TPZ and a much less toxic radical species. We have used yeast (Saccharomyces cerevisiae) as a model to better understand the mechanism of action of TPZ. Overexpression of NCP1, encoding the yeast ortholog of the human P450 oxidoreductase, results in greatly increased sensitivity to TPZ. Likewise, overexpression of TOP2 (encoding topoisomerase II) leads to hypersensitivity to TPZ, suggesting that topoisomerase II is also a target of TPZ in yeast. Thus, our data show that yeast mimics human cells in terms of TPZ sensitivity. We have performed robot-aided screens for altered sensitivity to TPZ using a collection of approximately 4600 haploid yeast deletion strains. We have identified 117 and 73 genes whose deletion results in increased or decreased resistance to TPZ, respectively. For example, cells lacking various DNA repair genes are hypersensitive to TPZ. In contrast, deletion of genes encoding some amino acid permeases results in cells that are resistant to TPZ. This suggests that permeases may be involved in intracellular uptake of TPZ. Our discoveries in yeast may lead to a better understanding of TPZ biology in humans.

Antineoplastic Agents↗
PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗
PubMed1986

Tandem regions of yeast DNA topoisomerase II share homology with different subunits of bacterial gyrase.

The nucleotide sequence for the Saccharomyces cerevisiae gene TOP2, which encodes DNA topoisomerase II, was compared with the sequence for bacterial DNA gyrase. The amino and carboxyl terminal halves of the single-subunit yeast enzyme showed homologies with the B and A subunits of bacterial gyrase, respectively, at corresponding positions along the polypeptide chains. Although the two enzymes differ in both quaternary structure and activity, the homology between the two proteins indicates mechanistic as well as structural similarities, and a probable evolutionary relationship.

Bacillus subtilis↗
PubMed2003

Sequence elements in both the intergenic space and the 3' untranslated region of the Crithidia fasciculata KAP3 gene are required for cell cycle regulation of KAP3 mRNA.

mRNA levels of several Crithidia fasciculata genes involved in DNA metabolism have previously been found to cycle as cells progress through the cell cycle. Octamer consensus sequences in the 5' untranslated regions (5' UTRs) of these transcripts were shown to be required for cycling of these mRNAs. The KAP3 gene encodes a kinetoplast histone H1-like DNA binding protein, and its mRNA levels cycle in parallel with those of the kinetoplast DNA topoisomerase (TOP2), dihydrofolate reductase-thymidylate synthase (DHFR-TS), and the large subunit of the nuclear single-stranded DNA binding protein (RPA1). KAP3 mRNA contains two octamer consensus sequences in its 3' UTR but none in its 5' UTR. Mutation of these octamer sequences was not sufficient to prevent cycling of a sequence-tagged KAP3 mRNA expressed from a plasmid. Mutation of an octamer sequence contained on the precursor transcript but not on the mRNA, in addition to mutation of the two octamer sequences in the 3' UTR, was necessary to abolish cycling of the mRNA. The requirement for a sequence not present on the mature mRNA indicates that regulation of the mRNA levels by the octamer sequences occurs at or prior to splicing of the transcript. Incompletely processed RNAs containing octamer sequences were also found to accumulate during the cell cycle when the mRNA levels were lowest. These RNA species hybridize to both the KAP3 coding sequence and that of the downstream drug resistance gene, indicating a lack of processing within the intergenic region separating these genes. We propose a cell cycle-dependent interference in transcript processing mediated by octamer consensus sequences as a mechanism contributing to the cycling of such transcripts.

3' Untranslated Regions↗
PubMed1992

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

Saccharomyces cerevisiae 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, homologous loci, SAM1 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, TOP1 and TOP2, do not affect the rate of recombination between the SAM 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 RAD1 excision repair gene, because both the rate of SAM ectopic gene conversion and the conversion tract length were reduced in rad1 top3 mutant cells compared with top3 mutants. These results suggest that a RAD1-dependent function is involved in the processing of damaged DNA that results from the loss of Top3 activity, targeting such DNA for repair by recombination.

Cell Division↗