A novel family of TRF (DNA topoisomerase I-related function) genes required for proper nuclear segregation.

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 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 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 TRF4/5 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.     

Sensitivity to camptothecin in Aspergillus nidulans identifies a novel gene, scaA + , related to the cellular DNA damage response

The anti-cancer drug camptothecin targets eukaryotic DNA topoisomerase I by trapping the covalent complex formed between the catalytically active enzyme and DNA. We are interested in identifying factors, other than topoisomerase I, that are involved in mediating cellular sensitivity to camptothecin. To this end, we have isolated eighteen mutants that are sensitive to camptothecin (sca) in the filamentous fungus Aspergillus nidulans and characterised one of them, sca299. The mutant sca299 is hypersensitive to camptothecin, and sensitive to several different mutagenic agents and to actinomycin D. Using temperature-sensitive mutations in genes that are known to regulate the cell cycle, we showed that the camptothecin sensitivity of the mutant sca299 is not affected by a mitotic block. The abnormal nuclear morphology observed in the sca299 mutant strain suggests that the germlings might be undergoing mitosis in the presence of unrepaired DNA damage, which would result in mitotic catastrophe. The hypersensitivity of the sca299 mutant to camptothecin does not result from elevated levels of topoisomerase I mRNA or from alterations in enzyme activity. Using DNA-mediated complementation of the sca299 mutant phenotype, the scaA+ gene was cloned. This gene encodes a 594-amino acid product; moderate structural similarity suggests that the scaA gene product may be related to the human nibrin gene which encodes a product involved in DNA double-strand break repair. Strains disrupted in the scaA gene were sensitive to the anti-topoisomerase I agent berberine, the DNA crosslinking agents mitomycin C and cis-platinum, and also to t-butyl hydroperoxide, which is an inducer of oxidative stress.     

Intrinsic 5'-deoxyribose-5-phosphate lyase activity in Saccharomyces cerevisiae Trf4 protein with a possible role in base excision DNA repair

In Saccharomyces cerevisiae, the base excision DNA repair (BER) pathway has been thought to involve only a multinucleotide (long-patch) mechanism (LP-BER), in contrast to most known cases that include a major single-nucleotide pathway (SN-BER). The key step in mammalian SN-BER, removal of the 5'-terminal abasic residue generated by AP endonuclease incision, is effected by DNA polymerase beta (Polbeta). Computational analysis indicates that yeast Trf4 protein, with roles in sister chromatin cohesion and RNA quality control, is a new member of the X family of DNA polymerases that includes Polbeta. Previous studies of yeast trf4Delta mutants revealed hypersensitivity to methylmethane sulfonate (MMS) but not UV light, a characteristic of BER mutants in other organisms. We found that, like mammalian Polbeta, Trf4 is able to form a Schiff base intermediate with a 5'-deoxyribose-5-phosphate substrate and to excise the abasic residue through a dRP lyase activity. Also like Polbeta, Trf4 forms stable cross-links in vitro to 5'-incised 2-deoxyribonolactone residues in DNA. We determined the sensitivity to MMS of strains with a trf4Delta mutation in a rad27Delta background, in an AP lyase-deficient background (ogg1 ntg1 ntg2), or in a pol4Delta background. Only a RAD27 genetic interaction was detected: there was higher sensitivity for strains mutated in both TRF4 and RAD27 than either single mutant, and overexpression of Trf4 in a rad27Delta background partially suppressed MMS sensitivity. The data strongly suggest a role for Trf4 in a pathway parallel to the Rad27-dependent LP-BER in yeast. Finally, we demonstrate that Trf5 significantly affects MMS sensitivity and thus probably BER efficiency in cells expressing either wild-type Trf4 or a C-terminus-deleted form.     

Chromatin determinants impart camptothecin sensitivity

Camptothecin‐induced locking of topoisomerase 1 on DNA generates a physical barrier to replication fork progression and creates topological stress. By allowing replisome rotation, absence of the Tof1/Csm3 complex promotes the conversion of impending topological stress to DNA catenation and causes camptothecin hypersensitivity. Through synthetic viability screening, we discovered that histone H4 K16 deacetylation drives the sensitivity of yeast cells to camptothecin and that inactivation of this pathway by mutating H4 K16 or the genes SIR1‐4 suppresses much of the hypersensitivity of tof1? strains towards this agent. We show that disruption of rDNA or telomeric silencing does not mediate camptothecin resistance but that disruption of Sir1‐dependent chromatin domains is sufficient to suppress camptothecin sensitivity in wild‐type and tof1? cells. We suggest that topoisomerase 1 inhibition in proximity of these domains causes topological stress that leads to DNA hypercatenation, especially in the absence of the Tof1/Csm3 complex. Finally, we provide evidence of the evolutionarily conservation of this mechanism.     

Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere

The TRF2-Rap1 complex suppresses non-homologous end joining and interacts with DNAPK-C to prevent end joining. We previously demonstrated that hTRF2 is a double strand telomere binding protein that forms t-loops in vitro and recognizes three- and four-way junctions independent of DNA sequence. How the DNA binding characteristics of hTRF2 to DNA is altered in the presence of hRap1 however is not known. Here we utilized EM and quantitative gel retardation to characterize the DNA binding properties of hRap1 and the TRF2-Rap1 complex. Both gel filtration chromatography and mass analysis from two-dimensional projections showed that the TRF2-Rap1 complex exists in solution and binds to DNA as a complex consisting of four monomers each of hRap1 and hTRF2. EM revealed for the first time that hRap1 binds to DNA templates in the absence of hTRF2 with a preference for double strand-single strand junctions in a sequence independent manner. When hTRF2 and hRap1 are in a complex, its affinity for ds telomeric sequences is 2-fold higher than TRF2 alone and more than 10-fold higher for telomeric 3′ ends. This suggests that as hTRF2 recruits hRap1 to telomeric sequences, hRap1 alters the affinity of hTRF2 and its binding preference on telomeric DNA. Moreover, the TRF2-Rap1 complex has higher ability to re-model telomeric DNA than either component alone. This finding underlies the importance of complex formation between hRap1 and hTRF2 for telomere function and end protection.     

The isolation and genetic analysis of V79-derived etoposide sensitive Chinese hamster cell mutants: two new complementation groups of etoposide sensitive mutants

Using a replica plating microwell method, three Chinese hamster V79-derived cell lines, designated ETO1, ETO2 and ETO3, which exhibit hypersensitivity to the non-intercalating topoisomerase II inhibitor etoposide have been isolated. Mutant lines ETO2 and ETO3 are cross-sensitive to the topoisomerase II inhibitors adriamycin and streptonigrin; however, neither mutant is sensitive to the topoisomerase I inhibitor camptothecin, the bifunctional alkylating agent mitomycin C, nor hydrogen peroxide. In contrast, ETO1 is cross-sensitive to camptothecin but displays only slight sensitivity to adriamycin, streptonigrin and hydrogen peroxide, and is not sensitive to mitomycin C. It has been established through extensive cell fusion studies that all three mutants are genetically distinct, and that ETO2 and ETO3 genetically complement all other known etoposide-sensitive Chinese hamster cell mutants (i.e., irs1, XR-1, xrs1, V3, BLM2, ADR1, ADR3, ADR4 and ADR5) thus defining two new complementation groups of etoposide sensitive mutants. Interestingly, the hybrids created by the fusion irs2TOR (thioguanine and ouabain resistant)xETO1 and the reciprocal cross ETO1TORxirs2 both exhibited a response to camptothecin intermediate with respect to V79 and ETO1. It has been hypothesised that this partial complementation may be the result of intragenic complementation and that both ETO1 and irs2 result from mutations in the gene XRCC8. This study indicates that cellular responses to topoisomerase II inhibitors are complex and hypersensitivity may result from mutations in many different genes.     

A C-terminal Myb extension domain defines a novel family of double-strand telomeric DNA-binding proteins in Arabidopsis

Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis.     

Defective Brca2 influences topoisomerase I activity in mammalian cells

The Chinese hamster cell mutant V-C8 is defective in the Brca2 gene (Kraakman-van der Zwet et al., 2002, Cell Biol.; 22: 669). Here we report that V-C8 cells were 10-fold more sensitive to camptothecin, an inhibitor of topoisomerase I, than the parental V79 cells. The level of the relaxation activity of topoisomerase I in nuclear extracts was also lower (4-fold) in V-C8 than V79 cells, in spite of the fact that the level of the topoisomerase I protein was the same in these cells. The survival of V-C8 cells in the presence of camptothecin, the sensitivity of V-C8 topoisomerase I to camptothecin, and the level of the relaxation activity in V-C8 nuclear extract were almost completely restored by transfection of V-C8 cells with the murine Brca2 gene or by the transfer of human chromosome 13 providing the BRCA2 gene. These results indicate that the observed changes in the topoisomerase I activity in V-C8 are due to the defective function of the Brca2 gene.     

Nuclear topoisomerase II levels correlate with the sensitivity of mammalian cells to intercalating agents and epipodophyllotoxins

We have investigated the biochemical basis for the hypersensitivity to intercalating agents and epipodophyllotoxins of a Chinese hamster cell mutant, ADR-1. More topoisomerase II-induced DNA strand breaks are accumulated by ADR-1 than by parental CHO-K1 cells following exposure to the intercalating agent amsacrine. Levels of induced DNA strand breaks correlate with cell killing. Topoisomerase II activity is elevated in ADR-1 cells as a consequence of an increased cellular level of topoisomerase II protein. We have studied the phenotype of cell hybrids generated by fusing parental and mutant cells. The hybrid ADR-1/CHO-K1 exhibits normal levels of resistance to amsacrine and expresses the lower, parental level of topoisomerase II. These results provide additional evidence that topoisomerase II mediates the cytotoxic action of intercalating agents and epipodophyllotoxins and suggest that the intracellular level of topoisomerase II is an important determinant of cellular sensitivity to these drugs. This has implications for antitumor therapy. ADR-1 cells provide a model system for studying the effects of topoisomerase II overproduction on cell proliferation and chromosome organization.     

TRF4 is involved in polyadenylation of snRNAs in Drosophila melanogaster

The Saccharomyces cerevisiae poly(A) polymerases Trf4 and Trf5 are involved in an RNA quality control mechanism, where polyadenylated RNAs are degraded by the nuclear exosome. Although Trf4/5 homologue genes are distributed throughout multicellular organisms, their biological roles remain to be elucidated. We isolated here the two homologues of Trf4/5 in Drosophila melanogaster, named DmTRF4-1 and DmTRF4-2, and investigated their biological function. DmTRF4-1 displayed poly(A) polymerase activity in vitro, whereas DmTRF4-2 did not. Gene knockdown of DmTRF4-1 by RNA interference is lethal in flies, as is the case for the trf4 trf5 double mutants. In contrast, disruption of DmTRF4-2 results in viable flies. Cellular localization analysis suggested that DmTRF4-1 localizes in the nucleolus. Abnormal polyadenylation of snRNAs was observed in transgenic flies overexpressing DmTRF4-1 and was slightly increased by the suppression of DmRrp6, the 3′-5′ exonuclease of the nuclear exosome. These results suggest that DmTRF4-1 and DmRrp6 are involved in the polyadenylation-mediated degradation of snRNAs in vivo.     

Saccharomyces cerevisiae DNA polymerase epsilon and polymerase sigma interact physically and functionally,suggesting a role for polymerase epsilon in sister chromatid cohesion

The large subunit of Saccharomyces cerevisiae DNA polymerase epsilon, Pol2, comprises two essential functions. The N terminus has essential DNA polymerase activity. The C terminus is also essential, but its function is unknown. We report here that the C-terminal domain of Pol2 interacts with polymerase sigma (Pol sigma), a recently identified, essential nuclear nucleotidyl transferase encoded by two redundant genes, TRF4 and TRF5. This interaction is functional, since Pol sigma stimulates the polymerase activity of the Pol epsilon holoenzyme significantly. Since Trf4 is required for sister chromatid cohesion as well as for completion of S phase and repair, the interaction suggested that Pol epsilon, like Pol sigma, might form a link between the replication apparatus and sister chromatid cohesion and/or repair machinery. We present evidence that pol2 mutants are defective in sister chromatid cohesion. In addition, Pol2 interacts with SMC1, a subunit of the cohesin complex, and with ECO1/CTF7, required for establishing sister chromatid cohesion; and pol2 mutations act synergistically with smc1 and scc1. We also show that trf5 Delta mutants, like trf4 Delta mutants, are defective in DNA repair and sister chromatid cohesion.     

Characterization of a camptothecin-resistant human DNA topoisomerase I in an in vitro system for Simian virus 40 DNA replication.

DNA topoisomerase I was required for bidirectional DNA replication in an in vitro system for Simian virus 40 (SV40) DNA replication with purified proteins in which the replication fork moved at the rate of 260 nucleotides/min on average. DNA topoisomerase I purified from camptothecin-resistant human lymphoblastoid cells, which confers high resistance of cellular DNA replication to camptothecin [Andoh, T., Ishii, K., Suzuki, Y., Ikegami, Y., Kusunoki, Y., Takemoto, Y. & Okada, K. (1987) Proc. Natl Acad. Sci. USA 84, 5565-5569], was characterized using this system. The activity of stimulating bidirectional DNA replication was comparable between two topoisomerase I from parental and resistant cells, i.e. in its dose-response relationship and in its time course for DNA synthesis. Camptothecin severely inhibited the leading as well as the lagging strand synthesis in the reaction containing the wild type topoisomerase I but not the mutant type topoisomerase I. The mutant type topoisomerase I was over 125-fold as resistant to camptothecin as the wild type topoisomerase I. These results are in good agreement with those on the sensitivity of cellular DNA synthesis to camptothecin in the resistant cells. These findings suggest that topoisomerase I is involved in cellular DNA replication as a swivelase and the mutation conferring camptothecin-resistance on the enzyme does not affect its functional efficiency in this system.     

Substitutions of Asn-726 in the active site of yeast DNA topoisomerase I define novel mechanisms of stabilizing the covalent enzyme-DNA intermediate

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of camptothecin. Recent reports of enzyme structure highlight the importance of conserved amino acids N-terminal to the active site tyrosine and the involvement of Asn-726 in mediating Top1p sensitivity to camptothecin. To investigate the contribution of this residue to enzyme catalysis, we evaluated the effect of substituting His, Asp, or Ser for Asn-726 on yeast Top1p. Top1N726S and Top1N726D mutant proteins were resistant to camptothecin, although the Ser mutant was distinguished by a lack of detectable changes in activity. Thus, a basic residue immediately N-terminal to the active site tyrosine is required for camptothecin cytotoxicity. However, replacing Asn-726 with Asp or His interfered with distinct aspects of the catalytic cycle, resulting in cell lethality. In contrast to camptothecin, which inhibits enzyme-catalyzed religation of DNA, the His substituent enhanced the rate of DNA scission, whereas the Asp mutation diminished the enzyme binding of DNA. Yet, these effects on enzyme catalysis were not mutually exclusive as the His mutant was hypersensitive to camptothecin. These results suggest distinct mechanisms of poisoning DNA topoisomerase I may be explored in the development of antitumor agents capable of targeting different aspects of the Top1p catalytic cycle.     

Multiple endonucleases function to repair covalent topoisomerase I complexes in Saccharomyces cerevisiae

Topoisomerase I plays a vital role in relieving tension on DNA strands generated during replication. However if trapped by camptothecin or other DNA damage, topoisomerase protein complexes may stall replication forks producing DNA double-strand breaks (DSBs). Previous work has demonstrated that two structure-specific nucleases, Rad1 and Mus81, protect cells from camptothecin toxicity. In this study, we used a yeast deletion pool to identify genes that are important for growth in the presence of camptothecin. In addition to genes involved in DSB repair and recombination, we identified four genes with known or implicated nuclease activity, SLX1, SLX4, SAE2, and RAD27, that were also important for protection against camptothecin. Genetic analysis revealed that the flap endonucleases Slx4 and Sae2 represent new pathways parallel to Tdp1, Rad1, and Mus81 that protect cells from camptothecin toxicity. We show further that the function of Sae2 is likely due to its interaction with the endonuclease Mre11 and that the latter acts on an independent branch to repair camptothecin-induced damage. These results suggest that Mre11 (with Sae2) and Slx4 represent two new structure-specific endonucleases that protect cells from trapped topoisomerase by removing topoisomerase-DNA adducts.     

The deubiquitinating enzyme Doa4p protects cells from DNA topoisomerase I poisons

DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of an enzyme-DNA covalent complex that is reversibly stabilized by the antitumor drug, camptothecin (CPT). During S-phase, collisions with replication forks convert these complexes into cytotoxic DNA lesions that trigger cell cycle arrest and cell death. To investigate cellular responses to CPT-induced DNA damage, a yeast genetic screen identified conditional tah mutants with enhanced sensitivity to self-poisoning DNA topoisomerase I mutant (Top1T722Ap), which mimics the action of CPT. Mutant alleles of three genes, DOA4, SLA1 and SLA2, were recovered. A nonsense mutation in DOA4 eliminated the catalytic residues of the Doa4p deubiquitinating enzyme, yet retained the rhodanase domain. At 36 degrees C, this doa4-10 mutant exhibited increased sensitivity to CPT, osmotic stress, and hydroxyurea, and a reversible petite phenotype. However, the accumulation of pre-vacuolar class E vesicles that was observed in doa4Delta cells was not detected in the doa4-10 mutant. Mutations in SLA1 or SLA2, which alter actin cytoskeleton architecture, induced a conditional synthetic lethal phenotype in combination with doa4-10 in the absence of DNA damage. Here actin cytoskeleton defects coincided with the enhanced fragility of large-budded cells. In contrast, the enhanced sensitivity of doa4-10 mutant cells to Top1T722Ap was unrelated to alterations in endocytosis and was selectively suppressed by increased dosage of the ribonucleotide reductase inhibitor Sml1p. Additional studies suggest a role for Doa4p in the Rad9p checkpoint response to Top1p poisons. These findings indicate a functional link between ubiquitin-mediated proteolysis and cellular resistance to CPT-induced DNA damage.