A Novel Family of TRF (DNA Topoisomerase I-Related Function) Genes Required for Proper Nuclear Segregation

Nucleic Acids Research, Jun 1996

We recently reported the identification of a gene, TRF4 (for DNA topoisomerase related function), in a screen for mutations that are synthetically lethal with mutations in DNA topoisomerase I (top1). Here we describe the isolation of a second member of the TRF4 gene family, TRF5. Overexpression of TRF5 complements the inviability of top1 trf4 double mutants. The predicted Trf5 protein is 55% identical and 72% similar to Trf4p. As with Trf4p, a region of Trf5p is homologous to the catalytically dispensable N-terminus of Topip. The TRF4/5 function is essential as trf4 trf5 double mutants are inviable. A trf4 (ts) trf5 double mutant is hypersensitive to the anti-microtubule agent thiabendazole at a semi-permissive temperature, suggesting that TRF4/5function is required at the time of mitosis. Examination of nuclear morphology in a trf4 (ts) trf5 mutant at a restrictive temperature reveals the presence of many cells undergoing aberrant nuclear division, as well as many anucleate cells, demonstrating that the TRF4/5function is required for proper mitosis. Database searches reveal the existence of probable Schizosaccharomyces pombe and human homologs of Trf4p, indicating that TRF4 is the canonical member of a gene family that is highly conserved evolutionarily.

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A Novel Family of TRF (DNA Topoisomerase I-Related Function) Genes Required for Proper Nuclear Segregation

Irene B. Castao 0 Sharon Heath-Pagliuso 0 Ben U. Sadoff 0 David J. Fitzhugh 0 Michael F. Christman 0 0 Department of Radiation Oncology, University of California , San Francisco, CA 94143, USA We recently reported the identification of a gene,TRF4 (for DNA topoisomerase related function), in a screen for mutations that are synthetically lethal with mutations in DNA topoisomerase I (top1). Here we describe the isolation of a second member of theTRF4 gene family, TRF5. Overexpression of TRF5 complements the inviability of top1 trf4 double mutants. The predicted Trf5 protein is 55% identical and 72% similar to Trf4p. As with Trf4p, a region of Trf5p is homologous to the catalytically dispensable N-terminus of Top1p. The TRF4/5 function is essential as trf4 trf5 double mutants are inviable. A trf4 (ts) trf5 double mutant is hypersensitive to the anti-microtubule agent thiabendazole at a semi-permissive temperature, suggesting that TRF4/5 function is required at the time of mitosis. Examination of nuclear morphology in atrf4 (ts) trf5 mutant at a restrictive temperature reveals the presence of many cells undergoing aberrant nuclear division, as well as many anucleate cells, demonstrating that the TRF4/5 function is required for proper mitosis. Database searches reveal the existence of probable Schizosaccharomyces pombe and human homologs of Trf4p, indicating that TRF4 is the canonical member of a gene family that is highly conserved evolutionarily. - Chromosomes are involved in dynamic cellular processes, such as DNA replication, transcription, chromatin assembly and genetic recombination, that lead to the formation of local domains of torsional stress (reviewed in 1). In Saccharomyces cerevisiae DNA topoisomerases I and II (topo I and II) appear to act together during DNA replication as a swivel to prevent the formation of positive supercoils ahead of the DNA replication fork. In S.cerevisiae under conditions where both topo I and II are inactivated, DNA replication stops rapidly (2), with elongation of new DNA chains continuing for only a few thousand nucleotides (3). Similar results have been obtained for Schizosaccharomyces pombe (4). * To whom correspondence should be addressed Transcription can also lead to the formation of locally supercoiled domains in DNA. In the twin domain model (5), movement of a transcription complex along the helical backbone generates positive supercoils ahead of the complex and negative supercoils behind the complex. It has been suggested that transcription is a major determinant of supercoiling in vivo (6). In S.cerevisiae, topo I and II appear to function together as a swivel for rRNA transcription and, to a lesser extent, for mRNA transcription (2). Transcription from a strong promoter can lead to hypernegative supercoiling of plasmids in top1 mutants (6,7), suggesting that topo I normally removes negative supercoils formed during transcription. These results and others have led to the suggestion that another major role for topo I is to relieve torsional stress generated during transcription. Despite the considerable evidence for the involvement of topo I in both DNA replication and transcription, topo I is not essential in either S.cerevisiae or S.pombe. Null mutations in the gene encoding the only type I DNA topoisomerase activity detectable in crude extracts cause only modest growth defects (4,8,9). Furthermore, the overall rates of both DNA and RNA synthesis are normal in top1 mutants (2), supporting the suggestion that topo II activity can substitute for topo I activity in these crucial processes (2,4,10). In S.pombe, conditional top2 mutants have been isolated that are inviable at the permissive temperature in combination with a top1 null mutation (11). In S.cerevisiae, top1 null mutants display a synthetic growth defect in combination with some top2 alleles (10) and we have identified alleles of TOP2 in a screen for top1 synthetic lethal mutations (12). These results demonstrate that some of the functions of type I and type II topoisomerases overlap. It remains unclear, however, whether topo I has additional functions distinct from those of topo II. The biological function of a second type I topoisomerase, topo III, (13,14), remains unclear. Null mutations in TOP3 result in hyper-recombination and slow growth (15,16), but top1 top3 double mutants are viable. Association of Top3p with Sgs1p, a DNA helicase, has been proposed to result in formation of a eukaryotic reverse gyrase (17). In order to investigate further the in vivo functions of topo I, we have perfomed a genetic screen to identify mutations affecting gene products that perform overlapping or dependent functions (18) with topo I and, thereby, to further elucidate which processes in the cell require topo I. We have identified four complementation groups of mutants with this phenotype called TRF, for DNA topoisomerase I-related function (12). The predicted Trf4 protein shares a region of homology with the N-terminus of Top1p. We report here the isolation of a TRF4 homolog, TRF5. DNA sequence analysis reveals that the predicted Trf4 and Trf5 proteins are 57% identical and 72% similar over their entire lengths. Overexpression of TRF5 complements the inviability of top1 trf4 double mutants. A trf4 (ts) trf5 double mutant is hypersensitive to the anti-microtubule agent thiabendazole at a semi-permissive temperature and grossly defective in nuclear division at a restrictive temperature, demonstrating that TRF4/5 function is required for proper mitosis. Database searches reveal the existence of probable S.pombe and human and homologs of TRF4/5, indicating that this novel gene family is evolutionarily conserved. MATERIALS AND METHODS Yeast strains (Table 1) were transformed using the lithium acetate method (19). The TRF5 gene was isolated from a genomic DNA library consisting of random S.cerevisiae DNA Sau3A fragments inserted into the BamHI site of YEp24. Escherichia coli cells (strain DH5a ) were transformed by electroporation (20). Small scale plasmid DNA preparations were made by the boiling lysis method (21) or using Qiagen columns (Qiagen, Chatsworth, CA). Yeast strain construction and crosses CY667. Strains CY726 and CY445 were crossed and sporulated. CY726. Derived from CY429 following EMS mutagenesis. CY855 and CY857. Plasmid pCB470 was digested with SacI to release a trf4-101::HIS3 fragment. This fragment was used to disrupt TRF4 in the TRF4/TRF4 diploid CY858. The resulting diploid was sporulated and several tetrads were dissected. One of the His+, cold-sensitive spores was designated CY855; another cold-sensitive His+ spore was designated CY857. Disruption at the TRF4 locus in CY855 was confirmed by Southern blot analysis. CY869. pCB469 was digested with NotI and XhoI to release trf4-102::TRP1 and the resulting fragment was used to disrupt TRF4 in CY184 to create CY869. CY870, CY872 and CY874. Plasmid pSH5 was digested with BamHI and PstI to release a trf5-3::LEU2 fragment. This fragment was used to disrupt TRF5 in the TRF5/TRF5 diploid (CY858). Disruption at the TRF5 locus at one allele was confirmed by Southern blotting. The resulting diploid (CY870) was sporulated and several tetrads were dissected. One of the Leu+ spores was designated CY874 (trf5-3::LEU2). A sister spore that is TRF5+ was designated CY872. CY908. Plasmid pSH5 was digested with BamHI and PstI to release a trf5-3::LEU2 fragment. This fragment was used to disrupt TRF5 in the trf4-101::HIS3/trf4-101::HIS3 homozygous diploid (CY892) to generate CY908. Disruption of TRF5 was confirmed by Southern blotting. The resulting diploid was sporulated and tetrads were dissected to test for synthetic lethality between the trf4 and trf5 mutations. MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 rDNA::URA3 MATa ade2-1 ura3-1 trp1-1 his3-11,15 leu2-3,112 rDNA::ADE2. MATa top1-7::LEU2 ade2-1 ura3-1 trp1-1 his3-11,15 leu2-3,112 rDNA::ADE2 MATa top1-7::LEU2 ade2-1 ura3-1 trp1-1 his3-11,15 leu2-3,112 ade3::hisG MATa top1-7::LEU2 ade2-1 ura3-1 his 3-11,15 trp1-1 leu2-3,112 ade3::hisG/pBS3 CY429 switched to MATa with pCB10. MATa top1-7::LEU2 trf4-1 his3-11,15 trp1-1 ade2-1 ade3/pBS3 MATa top1-7::LEU2 trf4-1 his3-11,15 trp1-1 ura3-1 ade2-1ade3::hisG MATa trf4-101::HIS3 ura3-1 ade2-1 his3-11,15 trp1-1 leu2-3,112 rDNA::ADE2 MATa trf4-101::HIS3 ura3-1 ade2-1 his3-11,15 trp1-1 leu2-3,112 rDNA::URA3 Diploid from CY184 X CY143 MATa trf4-102::TRP1 ura3-1 ade2-1 his3-11,15 trp1-1 leu2-3,112 rDNA::ADE2 Diploid CY858 disrupted with trf5-3::LEU2 at one allele MATa ura3-1 ade2-1 his 3-11,15 trp1-1 leu2-3,112 rDNA::ADE2 MATa trf5-3::LEU2 ura3-1 ade2-1 his3-11,15 trp1-1 leu2-112,3 rDNA::ADE2 Diploid from CY855 CY857 trf4-101::HIS3/trf-101::HIS3, TRF5+/trf5-3::LEU2 MATa trf4-101::HIS3 trf5-3::LEU2 ura3-1 ade2-1 his3-11,15 trp1-1 leu2-3,112 rDNA::ADE2/pCB432 MATa trf4-101::HIS3 trf5-3::LEU2 ura3-1 ade2-1 his3-11,15 trp1-1 leu2-3,112 rDNA::URA3/pCB639 (trf4-ts2) Media and growth conditions Yeast strains were routinely grown in YEP with 2% glucose (YPD). Thiabendazole (60 m g/ml) was added to YPD agar just prior to pouring the plates. Unless otherwise stated, cells were grown at 30 C. DNA sequencing and mapping A 3.0 kb fragment of genomic DNA containing TRF5 was cloned into YEplac112 and sequenced by the chain termination method (22) using a Sequenase kit (US Biochemicals, Cleveland, OH) and [35S]dATP. The 3.0 kb XhoIXhoI insert from pSH4 was sequenced on both strands using synthetic primers. To map TRF5, this fragment was radiolabeled and hybridized to filters containing l clones spanning the yeast genome (23). Plasmid constructions (Table 2) pSH2. pCB426 was digested with BamHI and the 10.6 kb fragment carrying TRF5 was ligated into the BamHI site of YEplac112. pSH3. pSH2 was digested with EcoRI and the 4.2 kb fragment was ligated into the EcoRI site of YEplac112. pSH4. pSH2 was digested with XhoI and the 3.0 kb fragment was ligated into the SalI site of YEplac112. pSH5. The LEU2 gene was excised from YEp13 by cutting with XhoI and SalI and ligated into the SalI site of pSH4. pSH6. pSH4 was digested with SalI and the ends were filled in with Klenow enzyme then religated to create a +4 frameshift (f.s.) mutation in TRF5. pCB432. The plasmid containing the library clone of TRF4 (pCB427) was digested with SnaBI and partially with HindIII to release a 3.7 kb fragment that was subsequently inserted into the HindIII (left) and SmaI (right) sites of pRS316. The resulting construct was designated pCB432. pCB469. pCB432 was digested with BamHI, the first 288 amino acids of Trf4p were removed and the TRP1 fragment was excised from pCB53 with a BamHI/BglII digest and ligated to the deleted TRF4 gene in pCB432, creating pCB469. pCB635. A PCR product containing TRF4 and 396 nt upstream of the ATG was cloned into the BamHI site of pRS314. pCB693. Identical to pCB635 (above) except that it carries the G317D mutation (trf4-ts2) in TRF4. Precise mapping of the TRF5 locus CY726 (top1-7::LEU2 trf4-1) carrying pBS3 (TOP1 URA3 ADE3 2u) was transformed with one of several subclones (pSH2, pSH3, pSH4, pSH5 or pSH6), grown non-selectively for the TOP1-containing plasmid (pBS3) and plated on 5-fluorotic acid (5FOA) at 30 C. 5-FOA medium provides a URA3+ counterselection, so that cells that are unable to lose pBS3 will not grow on this medium. The 10.6 kb fragment containing TRF5 (pSH2) permitted growth of Ura segregants, indicating that TRF5 complements the synthetic lethality of top1 trf4 double mutants in addition to the trf4-1 cs phenotype. The subclone containing a 4.2 kb EcoRI fragment (pSH3) did not complement. However, a plasmid containing the 3.0 kb XhoIXhoI fragment (pSH4) did, indicating that this fragment contained the TRF5 gene. TRF4 URA3 CEN (library) trf4101::HIS3 URA3 CEN trf4-ts2 (G317D) TRP1 CEN TOP1 ADE3 URA3 2u Isolation of a temperature-sensitive trf4 allele Plasmid pCB635 was mutagenized in vitro with hydroxylamine (24) and transformed into yeast strain CY924 (trf5 trf4/pTRF4.URA3) by selection for Trp+. Transformants were grown non-selectively for the pTRF4.URA3 plasmid and replica printed to 5-FOA plates at 24 and 37 C. Colonies able to give rise to 5-FOA-resistant segregants at 24 C but not at 37 C were identified. Plasmid pCB693 was recovered from 5-FOA-resistant segregants of trf4 ts candidates grown at 24 C and used to retransform CY924 to confirm that trf4 ts activity was plasmid associated. For one such isolate, the entire TRF4 open reading frame was sequenced. The only mutation found was a GA transition, resulting in a missense mutation of Gly317 to Asp (G317D or ts2). TRF5 overexpression complements a mutation in trf4 A 2m yeast genomic library was screened for plasmids that suppressed the cs phenotype of a trf4-1 mutant strain (CY667/pBS3) in order to clone the TRF4 gene. In addition to TRF4 (12), a clone containing a different 10.6 kb insert based on restriction mapping (pCB426) was identified. This gene was designated TRF5. Subsequently, TRF5 was found to complement the synthetic lethality between trf4 and top1 based on its ability to allow a top1 trf4 double mutant to survive following loss of a TOP1.URA3 plasmid and, thereby, to give rise to 5-FOA-resistant segregants (Fig. 1A). Complementation of top1 trf4 synthetic lethality was used to localize TRF5 to a 3.0 kb region (Fig. 1B). A SalI site within this 3.0 kb region was used to generate a LEU2 Figure 3. Homology between the Trf5p C-terminus and the Top1p N-terminus. The alignment was generated using the GENWORKS sequence analysis software from Intelligenetics. Boxed residues are those with a cost of 3. insertion mutation (pSH5) and a frameshift mutation allele (pSH6) in TRF5. Neither of these trf5 alleles complemented the inviability of a top1 trf4 double mutant (Fig. 1A). These results indicate that TRF5 is contained within the 3.0 kb XhoIXhoI fragment on pSH4. Hybridization of this region to filters containing DNA from contigs covering 90% of the yeast genome (23) revealed that TRF5 mapped to the left arm of chromosome XIV ~ 40 kb centromere distal to MET2. TRF4 and TRF5 define a novel gene family The 3.0 kb XhoIXhoI fragment containing TRF5 was sequenced on both strands (GenBank accession no. U47282). The predicted TRF5 open reading frame encodes a 625 amino acid protein (Fig. 2) whose sequence is 57% identical and 72% similar to Trf4p. A search of the GenBank database reveals the existence of a clear S.pombe homolog of TRF4/5 identified in the S.pombe genome project (Fig. 2). The predicted S.pombe homolog, called SPAC12G12.13c, is 33% identical and 56% similar to Trf4p over its C-terminal 683 amino acids. However, it also has a 570 amino acid N-terminal extension relative to Trf4p that is highly homologous to the G-b protein family and completely unrelated to Trf4p or Trf5p. The function of this protein in S.pombe is not known. Short regions of possible homology to Trf4p were also found in two human expressed sequence tags (ESTs; Fig. 2). The first human EST (accession no. H85548) encodes a predicted peptide of 131 amino acids (H85548.pep in Fig. 2) that is 41% identical and 60% similar to a region of Trf4p. The second EST (accession no. H90950) encodes a predicted 130 amino acid peptide (H90950.pep in Fig. 2) that is 44% identical and 65% similar to a different region of Trf4p. The two EST peptides overlap but are not identical at the DNA sequence level, indicating that they are probably derived from different genes. In addition, two short predicted peptides (59 and 24 amino acids) derived from a Drosophila STS (accession no. G01479) are 42% identical and 61% similar to Trf4p (not shown). This sequence may traverse an intron/exon boundary, since it not derived from cDNA. The function of these putative TRF4 homologs is not known. Examination of the Trf4p-related sequences with gcg programs such as MOTIFS and with BLOCKS motif databases also failed to identify meaningful signature regions. In addition, limited sequence homology was also found between a 91 amino acid N-terminal region of S.cerevisiae Top1p and a 92 amino acid C-terminal region of Trf5p (Fig. 3). These regions are 33% identical and 58% similar. The alignment, while limited, is likely to be significant as it is 6.06 SD above the mean significance of 20 randomized alignments of the same two sequences using the gcg sequence analysis program BESTFIT. We previously reported homology between the N-terminus of Top1p and the N-terminus of Trf4p (12). Thus, a Top1p-related region exists at the N-terminus of Trf4p, but at the C-terminus of Trf5p. The N-terminus of Top1p is not required for catalytic activity (25) and may be involved in some other aspect of topo I function. TRF4 and TRF5 define an essential function that is required at the time of mitosis A null allele of TRF5 was constructed by inserting LEU2 into TRF5 to make trf5-3::LEU2 (Fig. 1B). This construct was used to disrupt one allele of TRF5 in a trf4/trf4 diploid (CY892). Following sporulation, 40 tetrads were dissected. All tetrads segregated two viable and two inviable spores, and all viable spores were Leu, indicating that the trf4 trf5 double mutant is inviable (Fig. 4). Because trf4 shows synthetic lethality with top1 (12), we tested for a genetic interaction between trf5 and top1. However, in contrast to top1 trf4 double mutants, the top1 trf5 double mutant was viable (data not shown). To examine the basis of the defect in cells deficient in TRF4/5 function, we constructed a conditional allele of trf4 by in vitro mutagenesis with hydroxylamine (see Materials and Methods). DNA sequence analysis showed the trf4 ts allele to be a change of Gly317 to Asp. The mutation lies in a region conserved among TRF4, TRF5 and SPAC12G12.13c. The trf4 (ts) trf5 double mutant grows somewhat more slowly than the wild-type at 29.5 C, but arrests growth at 37 C (Fig. 5). At a semi-permissive temperature of 32 C, the trf4 (ts) trf5 double mutant displays an increased sensitivity to the anti-microtubule drug thiabendazole (Fig. 5), suggesting that the common TRF4/5 function may occur at the time of mitosis. ase I) gene products serve overlapping or dependent functions in S.cerevisiae, as the double mutant, but neither single mutant, is inviable (12). TRF5 was isolated based on its ability to complement the cold sensitivity of a trf4-1 point mutation and encodes a second member of the TRF4 family. TRF5 overexpression also complements the inviability of a top1 trf4 double mutant and the predicted Trf5 protein has a region that is homologous to the N-terminus of Top1p. Putative TRF4 homologs from S.pombe, Drosophila and human were identified in database searches. trf4 trf5 double mutants are inviable, demonstrating that TRF4/5 function is essential. A conditional trf4 (ts) trf5 mutant was constructed and shown to be hypersensitive to the microtubule poison thiabendazole when grown at a semi-permissive temperature, as well as grossly defective in nuclear division at a restricitve temperature, indicating that TRF4/5 function is required at the time of mitosis. Percentage of cells with each To determine whether TRF4/5 function is required at mitosis, a direct examination of nuclear morphology was performed on the trf4 (ts) trf5 double mutant following a shift to a restrictive temperature (37 C) for 3 h (Fig. 6). Three hours after a shift to a restrictive temperature (37 C), 50% of the trf4 (ts) trf5 cells that are undergoing nuclear division display an aberrant nuclear morphology in which the dividing nucleus is primarily within the mother cell (arrows in Fig. 6). This is similar to the so called cut phenotype observed in certain S.pombe mutants that are defective in nuclear division, in which nuclear division is stalled and the nucleus is unequally distributed between mother and daughter cells (4). In addition, anucleate and binucleate cells resulting from a defective nuclear division are observed at greater frequency in trf4 (ts) trf5 mutants than in the wild-type (Fig. 6, 37 C panel). Quantitation of the defects in nuclear divison showed that in trf4 (ts) trf5 mutants shifted to 37 C for 3 h, 15 times more anucleate cells and 18 times more cut-like cells are observed than in an otherwise isogenic wild-type strain (Table3). Some of the trf4 (ts) trf5 defects are apparent even at the permissive temperature. For example, cells of the trf4 (ts) trf5 mutant are considerably larger than TRF+ cells even at 24 C. In addition, the trf4 (ts) trf5 mutant shows six times more anucleate cells and six times more cut-like nuclei than the wild-type at 24 C. These results indicate that TRF4/5 function is required for proper nuclear division. We report the identification of a novel gene, TRF5, that is related to the S.cerevisiae TRF4 (DNA topoisomerase I-related function) gene product. The TRF4 and TOP1 (encoding DNA topoisomer Both Trf4p and Trf5p show a region of limited but significant homology to the catalytically dispensable N-terminus of Top1p. This region is not conserved among type I DNA topoisomerases a50% of the cells that are in nuclear divison. and its role in Top1p function is not known. The high percentage of both positively and negatively charged residues in these regions (average 35%) may indicate that their function is to mediate interaction with chromatin, as similar regions are commonly found in HMG proteins (26). The Trf4 family of proteins does not show significant overall homology with other proteins in the GenBank database (release 91) that would suggest a biochemical activity, with the exception of the S.pombe homolog, which shows a significant homology to G-b proteins via its 570 amino acid N-terminal extension. G-b family members encode proteins involved in many processes (27), including initiation of DNA replication (CDC4), chromosome separation (CDC20) and transcriptional repression (TUP1). A 40 amino acid repeated sequence containing consecutive Trp and Asp residues, termed the WD40 repeat, is found between five and eight times in most family members and appears to mediate proteinprotein interaction (28). TRF4/5 function is required at the time of mitosis for proper nuclear division. A critical and poorly understood event in the chromosome cycle that occurs at mitosis is chromosome condensation. Thus, a plausible hypothesis is that TOP1, TRF4 and TRF5 are involved in mediating mitotic chromosome condensation. This model is consistent with the proposed mitotic function for TRF4/5. In addition, null mutations in trf4 are inviable in combination with a mutation in SMC1 (29), the yeast homolog of XCAP-C (30), a Xenopus protein that is required for mitotic chromosome condensation in vitro (I. B. Castao and M. F. Christman, unpublished observations). Taken together, these results suggest that the TRF4 gene family may encode proteins that mediate mitotic chromosome structure in some manner. We thank Pius Brzoska and Nikki Levin for critically reading the manuscript and Pragati Bakshi for help in preparing the manuscript. I.B.C. was supported in part by a fellowship from the Organization of American States and S.H.P. was supported by an NIH training grant (CA09215) post-doctoral fellowship. This work was supported by National Institutes of Health grant R01GM46877 to M.F.C.


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Irene B. Castaño, Sharon Heath-Pagliuso, Ben U. Sadoff, David J. Fitzhugh, Michael F. Christman. A Novel Family of TRF (DNA Topoisomerase I-Related Function) Genes Required for Proper Nuclear Segregation, Nucleic Acids Research, 1996, 2404-2410, DOI: 10.1093/nar/24.12.2404