From the Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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Camptothecin is an antitumor agent that kills
cells by converting DNA topoisomerase I into a DNA-damaging poison.
Although camptothecin derivatives are now being used to treat tumors in a variety of clinical protocols, the cellular factors that influence sensitivity to the drug are only beginning to be understood. We report
here that two genes required for sister chromatid cohesion, TRF4 and MCD1/SCC1, are also required to repair
camptothecin-mediated damage to DNA. The hypersensitivity to
camptothecin in the trf4 mutant does not result from
elevated expression of DNA topoisomerase I. We show that Trf4 is a
nuclear protein whose expression is cell cycle-regulated at a
post-transcriptional level. Suppression of camptothecin
hypersensitivity in the trf4 mutant by gene overexpression resulted in the isolation of three genes: another member of the TRF4 gene family, TRF5, and two genes that may
influence higher order chromosome structure, ZDS1 and
ZDS2. We have isolated and sequenced two human
TRF4 family members, hTRF4-1 and
hTRF4-2. The hTRF4-1
gene maps to chromosome 5p15, a region of frequent copy number
alteration in several tumor types. The evolutionary conservation of
TRF4 suggests that it may also influence mammalian cell
sensitivity to camptothecin.
Camptothecin is a plant alkaloid derived from the Chinese tree
Camptotheca acuminata which was initially discovered to be active against murine leukemia (1). Interest in camptothecin was
heightened with the discovery in 1985 that the drug caused DNA damage
by specifically targeting DNA topoisomerase I (2). The drug was shown
to stabilize a covalent reaction intermediate in which DNA
topoisomerase I is linked via a tyrosine residue in the protein to the
3'-phosphoryl end of the broken DNA strand (3, 4). The reversible
single strand nick that results is thought to lead to cytotoxicity by
conversion into an irreversible lethal lesion, probably a double strand
break, upon encounter of the damaged DNA with a DNA replication fork
(5). If the resulting double strand break is not repaired, cell death results.
A rigorous demonstration that DNA topoisomerase I (TOP1) is
the sole target of camptothecin came from studies of camptothecin action in yeast. Camptothecin was shown to kill the yeast
Saccharomyces cerevisiae and to poison the action of yeast
TOP1 as it does in mammalian cells. Significantly, yeast
cells deleted for the TOP1 gene are completely
resistant to killing by the drug (6, 7). Furthermore, expression of
human topoisomerase I in the top1 deletion strain restores
sensitivity to killing by camptothecin (6, 7). Thus, camptothecin kills
both mammalian and yeast cells by turning TOP1 into a
DNA-damaging agent.
Yeast has proven to be an extremely useful system in which to study the
parameters affecting camptothecin-mediated cytotoxicity because of the
availability of well characterized mutants in DNA repair pathways and
facile genetic methods (7-9). One important insight was the
observation that defects in double strand break repair cause
hypersensitivity to killing by camptothecin (6, 7), supporting the
notion that a double strand break generated during DNA replication is a
major lethal lesion. Indeed, in yeast cells as in mammalian cells,
agents that inhibit DNA synthesis also greatly reduce
camptothecin-mediated killing (10). The RAD52 epistasis
group is required for double strand break repair in S. cerevisiae, and mutations in RAD52 cause marked
hypersensitivity to camptothecin. Defects in the DNA damage checkpoint
pathways which cause hypersensitivity to camptothecin in mammalian
cells also cause inviability in yeast cells expressing a
top1 mutation that mimics the effects of camptothecin (11).
In addition, sensitivity to camptothecin can be modulated through
altered expression of multidrug resistance-related efflux pumps such as
SNQ2 (12) or through dominant mutations in transcriptional
regulators of these pumps (13). Studies in yeast are likely to lead to
further insights into the mechanism of repair of this unusual type of DNA damage, provided the important pathways and genes are
evolutionarily conserved.
We have discovered that TOP1 functions, together with a
novel gene called TRF4 (for Topoisomerase
Related Function), in the process of mitotic
chromosome condensation (14, 15). Furthermore, we have shown that the
Trf4 protein is physically associated with Smc1p and Smc2p (14),
proteins that bind directly to chromosomes and cause condensation (16).
The Smc protein complex can alter DNA topology (17), which is likely to
explain the functional redundancy with TOP1. Recent evidence
from our laboratory shows that TRF4 is also required to
maintain a different aspect of higher order chromosome structure,
sister chromatid cohesion.1
Thus, two key aspects of higher order chromosome structure, sister cohesion and mitotic chromosome condensation, are mediated by Trf4p
most likely through its association with Smc1p (14).
Here we report the surprising observation that the trf4
mutant is also profoundly hypersensitive to killing by camptothecin. We
also show that mutation of another Smc1p-associated protein required
for sister chromatid cohesion, Mcd1p/Scc1p, also results in dramatic
camptothecin hypersensitivity. It is of great importance to understand
the molecular mechanisms employed to repair DNA damage caused by
camptothecin because DNA repair deficiencies are likely to affect
sensitivity of cancer cells to camptothecin (18). Specific tumors known
to be defective in the relevant repair pathway might be significantly
more responsive to camptothecin therapy (19). A detailed knowledge of
the repair mechanisms used in tumors could lead to the identification
of novel drug targets. Furthermore, because double strand breaks are
known to lead to genomic instability such as gene amplification (20), a
thorough knowledge of the means by which these breaks are repaired is
central to understanding tumorigenesis. Our data suggest that higher
order chromosome structure may be a critical determinant of
camptothecin toxicity.
Chemicals and Media--
Camptothecin was purchased from Sigma
and dissolved in dimethyl sulfoxide at a stock concentration of 4 mg/ml. Methyl methanesulfonate (MMS),2 hydroxyurea, and
nocodazole were purchased from Sigma. Plasmids and Strains--
The plasmids and yeast strains used in
this study are listed in Tables I and
II, respectively. All gel purifications
were performed using Qiagen columns (Qiagen, Chatsworth, CA). All yeast transformations were performed using lithium acetate in the presence of
single-stranded DNA (21). Escherichia coli cells (strain DH5
To construct CY1112, CB1007 was digested with BstEII (which
cuts once in the LEU2 gene) and the linear fragment gel
purified and used to transform CY855 to Leu+. The genomic structure of the resulting integrant was confirmed by PCR. Transformants were screened by fluorescence microscopy to detect a
galactose-dependent GFP signal and tested for
complementation of cold sensitivity of CY855. CY1114 was constructed as
described above by transformation of CY890 with linear CB1007. Control
strains were constructed by transformation of CY855 and CY890 with
linear, BstEII-digested pRS305 and selection on SC plates
lacking leucine (SC-leu).
To construct CY1115, CB1007 was digested with PmlI (which
cuts once in the TRF4 gene) and the linear fragment gel
purified and used to transform CY143. Transformants were selected on
SC-leu and screened for expression of TRF4-GFP by
fluorescence microscopy.
Drug Sensitivity Assays--
Equal numbers of cells of wild-type
(CY184 and VG-906-1A), trf4::HIS3 (CY1000), and
mcd1-1 (CY1229) strains were serially diluted
10-fold, and 5-µl aliquots were spotted on YPD plates containing 10 µg/ml camptothecin and buffered with 25 mM HEPES, pH 7.2. Control plates contained final concentrations of 0.25% dimethyl
sulfoxide and 25 mM HEPES, pH 7.2. Plates were incubated at
30 °C for 2 days. Sensitivity to MMS was assayed as described above
for wild-type (CY184) and trf4::HIS3 (CY855)
strains. Cells were spotted on YPD plates containing 0.025% MMS.
UV Sensitivity Assay--
Equal numbers of cells of wild-type
(CY184), trf4::HIS3 (CY1000), and rad3
(CY674) strains were plated on YPD plates and exposed to increasing
doses of UV irradiation using a Stratagene UV Stratalinker. Plates were
immediately wrapped in aluminum foil and incubated at 30 °C for 2 days. Colonies were counted, and percent viability was calculated based
on the number of colonies arising without exposure to UV irradiation.
DNA Topoisomerase I Assay--
To prepare crude protein
extracts, a single colony from wild-type (CY184),
trf4::HIS3 (CY855), and
top1::LEU2 (CY154) was resuspended in 30 µl of
SED (1 M sorbitol, 25 mM EDTA, 50 mM dithiothreitol), and spheroplasts were prepared by the
addition of 4 µl of zymolase (5 mg/ml in 1 M sorbitol).
The spheroplasts were pelleted, lysed by the addition of 12 µl of
yeast lysis buffer (20 mM Tris-HCl, pH 7.5, 0.5 M KCl, 10% glycerol, 1 mM Na2EDTA,
1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride), and incubated for 30 min on ice. Cell debris was removed by
centrifugation, and supernatant containing the crude protein extract
was collected. The protein concentration was determined using a Bio-Rad
assay. The extracts were diluted in buffer consisting of 100 mM Tris-HCl, 200 mM NaCl, 1 mM
EDTA, 5% glycerol to a final concentration of 5 mg/ml. DNA topoisomerase assays were performed using a standard relaxation assay
(TopoGEN, Columbus, OH) using 1 µl of diluted crude protein extracts.
Reactions were terminated at timed intervals by the addition of stop
buffer (TopoGEN) and analyzed by electrophoresis on 0.8% agarose gels
and ethidium bromide staining.
Complementation of Cold Sensitivity--
To assess whether the
TRF4-GFP fusion was functional, we tested complementation of
the cold-sensitive phenotype of a trf4::HIS3 strain by integration of the pGAL-TRF4-GFP construct.
Strains CY1112, CY1114, and control strains containing pRS305
integrated at the LEU2 locus were streaked on plates
containing glucose and plates containing galactose. Duplicate plates
were incubated at 17 °C for 6 days and 30 °C for 2 days.
Functional pGAL-TRF4-GFP candidates were identified on the
basis of galactose-dependent growth at 17 °C.
Fluorescence Microscopy--
To prepare cells for fluorescence
microscopy, cells were grown overnight in YPD or YP-Gal. Aliquots of
cells were removed, treated with Suppression of Camptothecin Hypersensitivity by Gene
Overexpression--
A yeast genomic library containing
Sau3A inserts in vector pSEY18 (URA3.2µ) was used to
isolate suppressors. Library DNA was isolated from E. coli
by Wizard Midicolumn (Promega) and used to transform
trf4::HIS3 (CY1000). Transformants were selected on SC-ura. After 3 days of incubation at 30 °C, approximately 270,000 total transformants were pooled, concentrated in 15% glycerol, and stored at The trf4 and mcd1-1 Mutants Are Hypersensitive to Killing by
Camptothecin--
An otherwise isogenic set of
trf4::HIS3and TRF4+ yeast strains was
constructed by single step gene replacement (25) and then tested for
sensitivity to the antitumor agent camptothecin. Killing by
camptothecin was monitored on Petri plates with or without 10 µg/ml
camptothecin incorporated into the agar (see "Experimental
Procedures").
As shown in Fig. 1, the
trf4::HIS3 mutant cells are roughly 3 orders of
magnitude more sensitive to killing by camptothecin compared with the
otherwise isogenic wild-type parent strain. In the absence of
camptothecin the wild-type and trf4::HIS3 mutant cells grow equally well. A similar test was performed on congenic mcd1-1 and MCD1+ yeast strains. The
MCD1/SCC1 gene product, like TRF4, is physically
associated with SMC1 and required for sister chromatid
cohesion (26, 27). Because MCD1/SCC1 is an essential gene,
the test was performed at the semipermissive temperature of 30 °C.
The mcd1-1 mutant was observed to be about 4 orders of magnitude more sensitive to camptothecin than the congenic parent strain (Fig. 1). The parent wild-type strains show slightly different camptothecin sensitivities because of their being derived from different strain backgrounds.
DNA Topoisomerase I Activity Is Not Elevated in the trf4
Mutant--
It is known that overexpression of DNA topoisomerase I in
yeast results in increased sensitivity to camptothecin-mediated killing
because of the presence of higher concentrations of topoisomerase I
enzyme (8, 9). Because TOP1 and TRF4 provide
overlapping functions in yeast (14, 28), we reasoned that deletion of TRF4 might result in a compensatory elevation in
TOP1 expression and thereby sensitize the trf4
mutant cells to camptothecin.
We tested this possibility directly by quantitatively measuring DNA
topoisomerase I activity in crude extracts from isogenic trf4::HIS3 and TRF4+ cells.
Topoisomerase I activity was measured by the ability to relax a
negatively supercoiled plasmid DNA substrate in the absence of ATP as
described under "Experimental Procedures." Under these conditions
neither topoisomerase II nor topoisomerase III activity is detected.
Protein extracts were prepared from single yeast colonies.
Approximately 5 µg of protein from each extract was added to a 20-µl reaction volume containing 1 µg of negatively supercoiled plasmid DNA. The negatively supercoiled substrate migrates faster than
the relaxed reaction product on a 0.8% agarose gel. The reactions were
incubated at 37 °C, and reaction progress was monitored by withdrawal of aliquots, addition of stop buffer containing EDTA, and
electrophoresis on 0.8% agarose gels to separate negatively supercoiled substrate from the fully relaxed reaction product.
Fig. 2 shows that the relaxation
reactions proceed with equivalent kinetics in the
trf4::HIS3 and TRF4+ extracts over a
40-min period. Both reactions are nearly complete after 40 min,
although some incompletely relaxed topoisomers can be observed in both extracts. Control extracts from a strain containing a top1
deletion or mock reactions containing no added cell extract (Fig. 2)
show no ability to relax the supercoiled substrate DNA. Therefore, the
hypersensitivity to killing by camptothecin in the trf4
mutant is not caused by overexpression of DNA topoisomerase I.
The trf4 Mutant Is Hypersensitive to Killing by MMS but Not UV
Irradiation--
We also tested for sensitivity to other DNA-damaging
agents: MMS, which is an alkylating agent that induces a wide range of DNA lesions, and UV irradiation, which creates intrastrand pyrimidine dimers, a type of DNA damage fundamentally different from those induced
by camptothecin and MMS. As shown in Fig.
3A, the trf4 mutant
is 2-3 orders of magnitude more sensitive to MMS than the isogenic
wild-type strain. But, as seen in Fig. 3B, the
trf4 mutant is not more sensitive to UV irradiation than the
wild-type strain, whereas a control rad3 mutant is
profoundly hypersensitive. We conclude that the trf4 mutant
is defective in the repair of a some types of DNA damage which are
induced by both camptothecin and MMS.
The Trf4 Protein Is Localized to the Nucleus during the S and
G2/M Phases--
Another explanation for the
hypersensitivity of the trf4 mutant to camptothecin is that
the TRF4 gene product might be required to repair
camptothecin-mediated DNA damage. Because camptothecin killing is
largely an S phase event (5, 10), we sought to determine whether the
Trf4 protein is present in the nucleus during S phase. To do this we
generated an in-frame gene fusion of GFP to the COOH
terminus of TRF4. Complementation experiments (see "Experimental Procedures") demonstrated that the fusion protein is
functional (data not shown).
A strain containing an integrated TRF4-GFP fusion expressed
from the native TRF4 promoter (CY1115) was examined by
fluorescence microscopy. As shown in Fig.
4, TRF4-GFP clearly localizes
to the nucleus in cells arrested at G1, S, and
G2/M phases of the cell cycle. The DAPI signal indicates
the position of the nucleus which is perfectly coincident with the
GFP signal in all cells examined. Examination of cells under
differential interference contrast confirms the expected morphology for
each cell cycle stage: unbudded in G1, small budded in S,
and large budded in G2/M. The GFP signal was
always observed to be significantly lower in G1 cells
relative to S or G2/M, indicating that the abundance of
Trf4p is cell cycle regulated.
To determine whether the cell cycle regulation of TRF4-GFP
is a post-transcriptional event we examined localization of the fusion
protein expressed constitutively at high levels from the GAL1 promoter (CY1112) and reexamined the cell
cycle-regulated abundance by quantitative fluorescence microscopy. The
results are summarized in Table III.
Even when TRF4-GFP transcription is constitutively expressed
at high levels, cells in G1 showed an intensity only 13%
of that observed during S phase. Cells in the G2/M phase
showed a signal that was 56% as strong as in S phase. Thus, a
post-transcriptional event is likely to mediate the cell cycle
regulation of Trf4p. TRF4 contains two potential
anaphase-promoting complex "destruction box" motifs (29, 30),
suggesting that it may be regulated through cell
cycle-dependent proteolysis.
Suppression of Camptothecin Hypersensitivity in the trf4 Mutant by
Gene Overexpression Identifies Three Additional Genes--
To
understand better the nature of camptothecin hypersensitivity in the
trf4 mutant we performed a screen for genes whose overexpression could restore a wild-type level of camptothecin sensitivity to TRF4-deficient cells. Suppression by gene
overexpression has historically been a fruitful way to identify genes
acting downstream in a pathway or functioning in a parallel pathway
(31).
We transformed a 2-µm genomic DNA library into a
trf4::HIS3 mutant host strain (CY1000), initially
selecting only for the DNA library marker gene URA3. 2-µm
plasmids are maintained at approximately 20-50 copies/cell and
consequently overexpress most of the genes present on the insert (31).
More than 200,000 transformants from the library were pooled, diluted,
and then replated on plates containing camptothecin. At a low
concentration of camptothecin wild-type cells are able to form healthy
single colonies, whereas trf4::HIS3 mutant cells
are killed (Fig. 5A). We
examined 460,000 trf4::HIS3 mutant cells
containing library plasmids for growth on the camptothecin plates
during several rounds of screening. Plasmid DNA was recovered from 133 plasmid-dependent suppressors and transformed into E. coli.
Diagnostic PCRs were used to eliminate those clones that contained the
TRF4 gene itself. One such clone, 16-2, is shown in Fig.
5B. Limited restriction mapping placed the remaining library clones into three classes, and a member of each class was sequenced. One class of suppressor carried inserts containing the
TRF4-related gene, TRF5 (15). This demonstrates
that at least two genes from the evolutionarily conserved
TRF4 gene family affect cellular sensitivity to
camptothecin. The two other suppressor classes represented separate
regions of chromosome XIII which encoded related genes called
ZDS1 (clone 23-2 in Fig. 5B) and ZDS2
(clone 26-2 in Fig. 5B). Suppression of the camptothecin
hypersensitivity in the trf4 mutant was observed to be
considerably stronger for ZDS2 than for ZDS1.
Both ZDS genes have been identified previously in other
genetic screens (variously called NRC1, ZDS1, and
OSS1), many of which were designed to identify products
important for chromosome structure or cell cycle progression (32-34),
although no molecular mechanism has yet been identified for their action.
Isolation of hTRF4-1 and hTRF4-2, Human Homologs of
TRF4/5--
With the knowledge that two yeast TRF4 family
members can profoundly affect cellular sensitivity to camptothecin
and the fact that TRF4 is the canonical member of an
evolutionarily conserved gene family, we sought to identify human genes
related to TRF4. The presence or absence of human
TRF4 expression in tumor cells may likewise affect tumor
cell sensitivity to camptothecin. Searches of the existing data bases
with yeast TRF4 revealed the presence of two human expressed
sequence tags (h90950 and h85548) with high homology to TRF4
over short regions. However, the data base contained only 396 base
pairs of sequence for h90950 and 382 base pairs from h85548. We
obtained the clones from which the limited DNA sequences were derived
from the ATCC archives and sequenced each clone in its entirety on both strands.
The h85548 clone contained an insert of 3842 base pairs with a
potential TRF4-related open reading frame of 517 amino acids (nucleotides 2-1152), containing a potential initiator methionine at
position 41, which includes all of the regions of high evolutionary conservation in the TRF4 gene family. Thus, h85548 is likely
to encode a full-length human TRF4 homolog. We have
designated this full-length human clone hTRF4-1
(GenBank accession number AF089896). S. cerevisiae TRF4 and
hTRF4-1 are 39% identical and 51% similar over
310 amino acids in the central region of both proteins, demonstrating a
very high degree of evolutionary and likely functional conservation (Fig. 6). For comparison, the human and
yeast DNA topoisomerase I gene products are 44% identical and 54%
similar, indicating that the TRF4 gene family has been
conserved to a similar degree. The hTRF4-1 gene
is also 49% similar and 35% identical to the S. cerevisiae
TRF5 gene over the same conserved region of 310 amino acids.
The h90950 clone contained an insert of 1479 base pairs with a
potential TRF4-related open reading frame of 381 amino acids (nucleotides 3-1145). Therefore, it is likely to represent only a
partial sequence. We have designated this human gene
hTRF4-2 (GenBank accession number AF089897). The
hTRF4-1 and hTRF4-2 gene
products are 65% identical and 72% similar over the entire length of
hTRF4-2 (381 amino acids). S. cerevisiae TRF4 and hTRF4-2 are
37% identical and 48% similar over 222 amino acids. The S. cerevisiae TRF5 gene is 48% similar and 34% identical to
hTRF4-2 over a 221-amino acid region. An
alignment of TRF4 family members from S. cerevisiae, human, Schizosaccharomyces pombe, and
Caenorhabditis elegans reveals a high degree of conservation
across many species (Fig. 6). Homology to the TRF4 family is
also evident in a number of additional expressed sequence tags derived
from mouse, rat, pufferfish, and Arabidopsis.
hTRF4-1 Maps to a Region of Frequent Copy Number Alteration in
Several Tumor Types--
A search of the GenBank STS data base with
the hTRF4-1 nucleotide sequence revealed a 100%
nucleotide sequence match over 200 base pairs with the Whitehead Genome
Project physical marker STS WI-6634. This STS maps to chromosome 5 between 16 and 18 centimorgans from the end of the short (or "p")
arm,3 placing
hTRF4-1 at cytogenetic location 5p15. We have
shown previously that this region of 5p is among the most common
regions amplified in small cell lung tumor cell lines (35) and in
primary small cell tumors (36). In addition, amplifications in this
region are frequently found in high grade ovarian tumors (37). The amplifications identified to date are large and contain many genes. Our
data will provide a first step toward determining whether hTRF4-1 contributes to tumorigenesis or affects
camptothecin sensitivity in tumors with 5p15 amplifications.
We have shown that S. cerevisiae cells deficient in
either TRF4 or MCD1/SCC1, genes required for
sister chromatid cohesion and mitotic chromosome condensation, are also
hypersensitive to DNA damage caused by the antitumor agent camptothecin
and MMS. The hypersensitivity to camptothecin killing in the
trf4 mutant does not result from elevated expression of DNA
topoisomerase I. The abundance of TRF4 is greatest during S
phase, the cell cycle phase at which cells are most readily killed by
camptothecin and the likely point at which sister chromatid cohesion is
established. Suppression of camptothecin hypersensitivity in the
trf4 mutant by gene overexpression resulted in the isolation
of an additional S. cerevisiae member of the TRF4
gene family, TRF5, which is 55% identical and 72% similar
to TRF4. Two genes that may influence higher order
chromosome structure, ZDS1 and ZDS2, were also
isolated. To begin to determine whether TRF4 function in
tumor cells also influences camptothecin sensitivity we have isolated
and sequenced two human TRF4 family members,
hTRF4-1 and hTRF4-2. The
hTRF4-1 gene maps to 5p15, a region that is
frequently amplified in a variety of tumor types. The high degree
of evolutionary conservation of the TRF4 family suggests
that these proteins are likely to function similarly in all eukaryotes,
and therefore amplifications of the hTRF4-1 gene
in tumor cells may alter sensitivity to camptothecin in these tumors.
The primary lethal lesion generated by camptothecin is likely to be a
replication-coupled double strand break (8). Two known repair pathways
are important in the repair of DNA double strand breaks: homologous
recombination and direct joining of nonhomologous ends. Mutations in
the homologous recombination pathway cause dramatic hypersensitivity to
MMS, a phenotype also observed in the trf4 mutant. The
trf4 mutant is not hypersensitive to UV irradiation, which
suggests that chromosome structure is not grossly altered such that all
repair is inhibited. We have shown that Trf4p is physically associated
with Smc1p (14). It may be relevant that Smc proteins have been shown
to catalyze homologous strand synapsis in vitro as
efficiently as the recA protein (38). We have demonstrated
that TRF4/TRF5 function is required for sister chromatid
cohesion.1 The presence of Trf4p on chromosomes when
cohesion is being established during S phase may facilitate its role in
repair of double strand breaks. Thus, TRF4 might be required
for recombinational repair of camptothecin damage. Alternatively,
TRF4 and MCD1/SCC1 may function in DNA repair but
not as part of an Smc protein complex.
The proteins encoded by the trf4 suppressors ZDS1
and ZDS2 are 38% identical over their nearly 1,000 amino
acid lengths but do not otherwise contain informative primary sequence
motifs. Screens in which these genes were identified previously include those designed to enhance replication origin function (34), to suppress
defects resulting from histone mutations
(33),4 and to suppress
mutations in CDC20, a gene required for chromosome segregation.5 It has been
suggested that these genes may have been found in different screens
because of their ability to alter chromosome structure globally (34).
This is consistent with the notion that higher order structure may be
an important and largely unrecognized determinant of camptothecin toxicity.
Yeast has proven to be a powerful system to study the targets and
repair of DNA damage caused by camptothecin and other antitumor agents
(7-9). The fact that evolutionarily conserved cohesion proteins are
required to repair DNA damage caused by camptothecin in yeast suggests
that this will also be true in mammalian cells. The advent of clinical
protocols that include camptothecin heightens the importance of
understanding those cellular functions that affect cytotoxicity. Future
studies on the functions of the human TRF4 genes
hTRF4-1 and hTRF4-2, whose
isolation is reported here, will address this directly.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-Factor was purchased from
Fluka. Yeast strains were routinely grown in YEP or synthetic complete
(SC) medium lacking the appropriate supplements with a 2% final
concentration of glucose (YPD) or galactose (YP-Gal).
) were transformed by electroporation (22). To construct CB1001,
pRS305 was digested with HindIII and PstI, and
the linear backbone was gel purified and ligated to a 736-base pair
HindIII/PstI fragment containing green
fluorescence protein (GFP) excised from plasmid
yGFP. Primers containing SalI restriction sites
and complementary to the TRF4 gene and a region upstream of
the GAL1 promoter on CB636, respectively, were used to
generate a Pfu DNA polymerase PCR product containing the
GAL1 promoter fused to the TRF4 gene with
SalI ends (pGAL-TRF4). This PCR product was
purified, digested with SalI, and gel purified. To construct
CB1007, CB1001 was digested with SalI, treated with calf
intestinal alkaline phosphatase, and the linear fragment was then gel
purified and ligated to SalI-digested pGAL-TRF4
PCR product.
Plasmids used in this study
Yeast strains used in this study
-factor (25 µg/ml final
concentration), hydroxyurea (100 mM final concentration),
or nocodazole (15 µg/ml final concentration), and incubated for
3 h at 30 °C in the presence of drug. Arrested cells were fixed
with 70% ethanol and treated with 0.2 µg/ml DAPI in 0.85% saline
for 45 min at room temperature in the dark. Cells were diluted in
mounting solution (0.1% r-phenylenediamine, 1 × phosphate-buffered saline in 90% glycerol) and placed on glass slides.
Cells were examined using a Nikon Eclipse 800 microscope with
differential interference contrast optics and DAPI and fluorescein isothiocyanate filters. Images were captured digitally using a Princeton Instruments CCD camera with IP Lab spectrum software and
colorized using Adobe Photoshop. Relative fluorescence intensities were
determined using a Chroma GFP filter and IP Lab spectrum software.
70 °C. Aliquots of transformants were periodically thawed, and the cells were diluted and plated on camptothecin plates.
To prepare camptothecin plates, 375 µl of a camptothecin stock
solution at 4 mg/ml in dimethyl sulfoxide was spread on YPD plates
buffered with 25 mM HEPES, pH 7.2, and allowed to dry. 460,000 library transformants were screened in this way.
Camptothecin-resistant colonies were identified after 2 and 3 days of
incubation at 30 °C. A 5-fluoro-orotic acid counterselection (23)
was used to isolate rare segregants that had spontaneously lost the
URA3-marked library plasmid, and the cells with no plasmid were then
retested for camptothecin resistance. 133 of the 166 candidate
suppressors were plasmid-dependent for the
camptothecin-resistant phenotype. Library plasmids were rescued from
yeast cells by "smash-n-grab" (24) and isolated from E. coli using Qiagen columns. Candidate clones were analyzed by PCR
using primers to TRF4, and those clones found to contain the
TRF4 gene were not analyzed further. Restriction digests of
the remaining plasmids revealed the presence of three different library
clones that were then sequenced using primers CP206 and CP207,
complementary to the multicloning sequence of pSEY18. The primer
sequences are: CP206, 5'-CGAATTCGAGCTCGGTACCCG-3'; CP207,
5'-TGCATGCCTGCAGGTCGACTC-3'.
RESULTS
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Fig. 1.
trf4 and
mcd1-1 mutants are hypersensitive to
killing by camptothecin. Equal numbers of cells from
trf4 (CY1000), wild-type (CY184 and VG906-1A), and
mcd1-1 (CY1229) strains were serially diluted
10-fold and then spotted on plates containing camptothecin
(CPT) and control plates containing no drug and incubated at
30 °C for 2 days. The wild-type strains for trf4 and
mcd1-1 are from different genetic backgrounds and
show slightly different camptothecin sensitivity.
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Fig. 2.
DNA topoisomerase I activity is not elevated
in trf4 relative to wild-type strains. DNA
topoisomerase I activity was examined in
trf4::HIS3 (CY855, TRF4 ) and wild-type (CY184,
TRF4+) crude protein extracts using a standard relaxation assay
(TopoGEN). Reactions were terminated at timed intervals and analyzed by
gel electrophoresis. Control lanes include the same substrate with
top1-7::LEU2 (CY154) extract and no
extract.
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Fig. 3.
The trf4 mutant is
hypersensitive to killing by MMS but not UV irradiation.
Panel A, equal numbers of cells from wild-type (CY184) and
trf4 (CY1000) strains were serially diluted 10-fold and then
spotted on plates containing MMS or no drug and incubated at 30 °C
for 2 days. Panel B, equal numbers of cells from wild-type
(CY184), trf4 (CY1000), and rad3 (CY674) strains
were plated on YPD and exposed to increasing doses of UV irradiation.
Colonies were counted after incubation at 30 °C for 2 days, and the
percent viability was calculated.
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Fig. 4.
Trf4-GFP protein is nuclear and cell
cycle-regulated. Cells containing TRF4-GFP under
control of the native TRF4 gene promoter (CY1115) were
arrested in G1, S, and G2/M phases of the cell
cycle and examined for GFP signal by fluorescence
microscopy. Images captured with the differential interference contrast
filter show cell morphology, the DAPI filter shows DNA, and the
fluorescein isothiocyanate (FITC) filter was used to
visualize the GFP signal. The efficacy of cell cycle arrest
was determined by examining cell morphology for 50 cells at
each arrest point.
Quantitation of TRF4-GFP signal under galactose induction
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Fig. 5.
ZDS1 and ZDS2
overexpression can suppress the camptothecin hypersensitivity of
a trf4 mutant. A 2-µm-based yeast genomic
library was introduced into a trf4::HIS3 strain
(CY1000), and rare camptothecin-resistant transformants were isolated.
The suppressing library plasmids were isolated and analyzed by PCR,
restriction digest, and sequencing. The genes identified include
ZDS1 and ZDS2. Panel A,
hypersensitivity of the trf4::HIS3 mutant to
camptothecin under the conditions of the screen. Panel
B, suppression of camptothecin hypersensitivity in
trf4::HIS3 by overexpression of TRF4
(16-2), ZDS1 (23-2), and ZDS2 (26-2).
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Fig. 6.
Alignment of the TRF4 gene
family. An alignment among six members of the TRF4 gene
family was made using the Wisconsin sequence analysis package program
"Pileup," and the alignment was formatted using the program
"Alscript." The order of alignment from top to
bottom is hTRF4-2,
hTRF4-1, S. cerevisiae TRF4, S. cerevisiae TRF5, S. pombe TRF4; and C. elegans
TRF4. The amino acid number corresponding to the S. cerevisiae TRF4 sequence is shown at the right of each
row.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Dan Burke, Vincent Guacci, Doug Koshland, M. Mitchell Smith, and Alex Strunnikov for strains and plasmids and M. Mitchell Smith and David Pellman for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the National Institutes of Health (to M. F. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Microbiology,
Box 441, Jordan Hall, 1300 Jefferson Park Ave., University of Virginia,
Charlottesville, VA 22908. Tel.: 804-243-2777; Fax: 804-982-1071;
E-mail: mfc3f{at}virginia.edu.
1 I. B. Castaño and M. F. Christman, unpublished observation.
3 Eric Lander and Thomas James Hudson, unpublished data.
4 M. Smith, personal communication.
5 Dan Burke, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: MMS, methyl methanesulfonate; SC medium, synthetic complete medium; PCR, polymerase chain reaction; DAPI, 4,6-diamidino-2-phenylindole.
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REFERENCES |
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