| Nucleic Acids Research | Pages |
New yeast genes important for chromosome integrity and segregation identified by dosage effects on genome stability
Introduction
Materials And Methods
Yeast strains, media and procedures
Colony color sectoring assay
Identification of CST clones
Estimation of the number of CST genes
Quantification of mitotic recombination frequency
Dosage interaction of CST genes with checkpoint mutations
Sensitivity to growth inhibitors
Deletions of CST genes
Results
Genes with dosage effect on chromosome stability
Cellular functions affected by CST overexpression
CST gene deletions
Human genes that affect chromosome stability in yeast
Discussion
Parameters and specificity of the screen
Phenotypes produced by overexpression of CST genes
Deletions of CST genes
Evolutionary conservation of CST genes
Acknowledgements
References
New yeast genes important for chromosome integrity and segregation identified by dosage effects on genome stability
Received June 10, 1999; Accepted June 18, 1999
ABSTRACT Phenotypes produced by gene overexpression may provide important clues to gene function. Here, we have performed a search for genes that affect chromosome stability when overexpressed in the budding yeast Saccharomyces cerevisiae. We have obtained clones encompassing 30 different genes. Twenty-four of these genes have been previously characterized. Most of them are involved in chromatin dynamics, cell cycle control, DNA replication or mitotic chromosome segregation. Six novel genes obtained in this screen were named CST (chromosome stability). Based on the pattern of genomic instability, interaction with checkpoint mutations and sensitivity to chromosome replication or segregation inhibitors, we conclude that overexpression of CST4 specifically interferes with mitotic chromosome segregation, and CST6 affects some aspect of DNA metabolism. The other CST genes had complex pleiotropic phenotypes. We have created deletions of five genes obtained in this screen, CST9, CST13, NAT1, SBA1 and FUN30. None of these genes is essential for viability, and deletions of NAT1 and SBA1 cause chromosome instability, a phenotype not previously associated with these genes. This work shows that analysis of dosage effects is complementary to mutational analysis of chromosome transmission fidelity, as it allows the identification of chromosome stability genes that have not been detected in mutational screens.
INTRODUCTION
Accurate propagation of genetic information requires the co-ordinated function of many cellular mechanisms. These include replication of the genome, detection and repair of spontaneous DNA damage and segregation of the duplicated and condensed chromosomes by the mitotic spindle. All of these events depend on proper control of chromatin architecture. High fidelity of these events is assured by monitoring and control by cell cycle regulatory mechanisms. Thus the genes important for maintenance of genome stability include the components and regulators of chromatin, DNA replication and repair machinery, mitotic apparatus components and cell cycle controls.
A large number of genes that affect chromosome stability in budding yeast have been identified by mutation screens (1-5). Additionally, many genes identified by other criteria were shown to be necessary for genome stability. The examples include DNA metabolism enzymes, such as polymerases and a ligase (6,7), and components of chromosome segregation machinery, including tubulins, mitotic spindle motors, components of the kinetochore, and spindle pole bodies (8-11).
In studies of mutations that affect chromosome stability, some genes important for this process may have been missed, either because their functions are redundant, or because only dominant mutations would have a phenotype. Genes affected by dominant mutations are difficult to clone because individual libraries are required. In this work, we broaden the mutational analysis of chromosome stability by the use of a dominant genetic approach.
Many of the events critical for genomic stability are known to be dependent on proper stoichiometric ratios of the components involved. It was shown, for example, that altered levels of histones or tubulins affect the fidelity of chromosome segregation, presumably by interfering with chromatin assembly and mitotic spindle functions, respectively (8-12).
Overproduction or inappropriate production of a normal gene product can affect cellular functions by different mechanisms. For components of multisubunit assemblies, an excess of one subunit can interfere with the formation of an active stoichiometric complex (dominant-negative effect). In this case, the phenotype produced by overexpression of the gene is similar to that of loss of function of the same gene. Numerous examples of this mechanism have been documented, including genes involved in chromosome segregation (3,8,13-15). Alternatively, the elevated amount of a protein can increase the activity of a normal cellular process, which might in itself lead to a detectable phenotype. This allows the identification of genes involved in maintenance of genomic stability by the chromosome loss phenotype resulting from their overexpression (15-17). Examples of successful overexpression-based screens include identification of proteins required for meiosis in budding yeast, and for mitotic cell division and cell morphogenesis in fission yeast (16,18,19). In particular, MIF2, a gene important for centromere function, and a possible homolog of human centromeric protein CENP-C, has been isolated and characterized through an overexpression-based screen (15,17). Overexpression has also been used successfully to pinpoint specific functional interactions between components of the kinetochore, and between proteins involved in DNA replication (20). In this work, we have exploited this approach to systematically search for genes important for chromosome stability (CST genes) in the budding yeast Saccharomyces cerevisiae.
MATERIALS AND METHODS
Yeast strains, media and procedures
The genotypes of the yeast strains used in this work are listed in Table 1. Strain YIIL39 was obtained by one-step gene replacement of MAD1 with HIS3, using pKH149 (provided by K. Hardwick and A. Murray). YIL40 was produced by replacing a 450 bp fragment of MAD3 with LEU2, using pKH515 (provided by K. Hardwick and A. Murray). Strain YF451 was obtained by backcrossing the mad2-1 allele into the S288C background (F. Spencer, personal communication).
Table 1. Yeast stains used in this study
| Strain | Genotype | Source |
| YPH275 | MATa/MAT[alpha] ura3-52 lys2-80 ade2-101 trp1-[Delta]1 his3-[Delta]200 leu2-[Delta]1 CF [TRP1 SUP11 CEN4] | P. Hieter |
| LH33OSD | MAT[alpha] ade2 leu2 trp1 hom3 ura3 can1 fcy1 sap3 (lys5 + cyh2 CEN7 ade6 +)/(+ aro2 + CEN7 + ade3) | L. Hartwell |
| TWY308 | MATa mec1-1 ura3 trp1 | T. Weinert |
| TWY398 | MATa [Delta]rad9::LEU2 his7 ura3 leu2 trp1 | T. Weinert |
| YIL39 | MATa [Delta]mad1::HIS3 ura3-52 lys2-801 ade2-101 trp1-[Delta]1 his3-[Delta]200 leu2-[Delta]1 CF [TRP1 SUP11 CEN4] | This study |
| YF451 | MATa mad2-1 ura3-52 lys2-801 ade2-101 his3-[Delta]200 trp1-[Delta]63 leu2-[Delta]1 CF [URA3 SUP11 CEN3] | F. Spencer |
| YIL40 | MATa [Delta]mad3::LEU2 ura3-52 lys2-801 ade2-101 trp1-[Delta]1 his3-[Delta]200 leu2-[Delta]1 CF [TRP1 SUP11 CEN4] | This study |
Colony color sectoring assay
Colony color sectoring assay was performed as described by (21), using the diploid yeast strain YPH275 (2) (Fig. 1). The frequency of loss and non-disjunction of the chromosome fragment (CF) was quantified by determining the frequency of half-red colonies within a population of pink sectoring colonies.
Figure 1. Colony color sectoring assay for chromosome loss (2;21). (A). The mechanism of colony color changes resulting from loss or gain of the CF. (B) An example of sectoring phenotype induced by overexpression of CST genes. Control cells (left) and cell overexpressing CST1/UBI2 (right), which shows an intermediate level of chromosome loss (10-fold increase over parental cells, compare to data in Table 3).
Identification of CST clones
We used a yeast cDNA library expressed from the GAL1 promoter in a CEN (single copy) vector (22). This library is derived from cells logarithmically growing in rich medium. The cDNA representation is estimated to be ~50-fold larger than the mRNA population (22). The library was transformed into the chromosome loss tester strain YPH275, and the cells were allowed to recover in liquid SC-Ura for 4 h. Transformants were plated onto SC-Ura containing 2% galactose to induce the expression of cDNA and a reduced concentration of adenine (5 mg/l) to facilitate red color development by ade2 cells. Cells were plated at a low density (500-1000 transformants per 150 mm plate). After 10 days at 30°C the plates were scored for the presence of sectored colonies (Fig. 1B). Colonies showing consistent sectoring phenotype on galactose, but not on glucose, were selected. Plasmids were rescued in Escherichia coli and re-transformed into YPH275. The inserts of the plasmids, which consistently induced galactose-dependent chromosome loss, were compared to each other by restriction mapping and Southern hybridization. The identity of unique inserts was established by partial sequencing.
Estimation of the number of CST genes
To estimate the minimal number of yeast genes that may have dosage effects on chromosome stability (CST-type genes), we used a simplifying assumption that all genes are equally represented in our cDNA library. In reality, unequal representation of cDNA species means that the number of CST genes is greater than estimated by this method. Given the equal representation assumption, the probability Px of recovering any gene exactly x times will be described by the Poisson distribution:
| Px = mx/x!em | 1 |
where m is the Poisson parameter. The observed frequency of obtaining a particular gene x times should be proportional to the probability Px of this event. If nx is the number of genes recovered x times (e.g. n0 genes were not encountered in this screen, n1 genes were recovered once each, n2 genes twice each, etc.), then
|
n0/n1 = P0/P1, nl/n2 = P1/P2, etc. |
2 |
Substituting the values of P0, P1 and P2 from 1 into 2 gives
|
n0/n1 = 1/m and n1/n2 = 2/m |
3 |
Thus n0, the number of CST genes missed by this screen, can be estimated from n1 and n2, the numbers of genes recovered once and twice each, respectively:
| n0 = n12/2n2 | 4 |
Since the abundance of different cDNAs in the library is not equal, this estimate provides a lower limit for the number of possible CST genes.
Quantification of mitotic recombination frequency
The frequency of mitotic recombination was determined as described (12), after introducing CST-overexpressing plasmids into the strain LH33OSD (Table 1). This is a haploid strain with an extra copy of chromosome VII. One of the copies of chromosome VII has an ade3 mutation on the right chromosome arm, and the other has ade6 on the right arm and a recessive cycloheximide resistance marker cyh2 on the left arm. This strain is also mutant for ade2 and therefore forms red colonies. If it becomes hemizygous or homozygous for ade3 and/or ade6, due to recombination, gene conversion or chromosome loss, the colony color changes to white. Cells that lose the wild-type CYH2 cycloheximide-sensitivity allele byloss can be selected on cycloheximide-containing plates. The mechanism of the ade3 and/or ade6 marker loss is then determined by the color of the colony, with chromosome loss producing white colonies, and recombination or gene conversion, red colonies (12). The frequency of mitotic recombination between cyh2 and the pluscentromere, plus the frequency of gene conversion at the CYH2 locus are thus proportional to the frequency at which red cycloheximide-resistant colonies arise (12).
Dosage interaction of CST genes with checkpoint mutations
CST-expressing plasmids were introduced into checkpoint mutant strains and the corresponding isogenic checkpoint-proficient strains. The cells were streaked on SC-Ura or SC, Gal-Ura plates, incubated at 30°C for 3-5 days (SC-Ura) or 7-14 days (SC, Gal-Ura), and scored for independent colony formation and colony size.
Sensitivity to growth inhibitors
Haploid cells containing CST-expressing plasmids or the control parental plasmid were streaked to single colonies on SC-Ura or SC, Gal-Ura plates, supplemented with the growth inhibitors in the following concentrations: 2.00-2.25 µg/ml methylmethane sulfonate (MMS); 125-175 mM hydroxyurea (HU); 12-17 µg/ml benomyl. The plates were incubated for 6-8 days at 30°C (HU and MMS) or for 10-12 days at 23°C (benomyl), and scored for independent colony growth and colony size.
Deletions of CST genes
Disruption fragments were generated by PCR amplification of the KanMX4 G418-resistance module, using the appropriate gene-specific primers with 40-50 bp identity to the corresponding gene at their 5[prime] ends (23). The resulting PCR fragments were transformed into YPH275, and G418-resistant colonies were selected. Correct replacement was verified by PCR amplification of the two replacement junction regions. Heterozygous diploid cells were sporulated and gene disruption was verified in haploid progeny by G418 resistance and PCR.
RESULTS
Genes with dosage effect on chromosome stability
We have performed a search for genes that affect chromosome stability when overexpressed in budding yeast S.cerevisiae. We used a library of S.cerevisiae cDNA in a centromere-containing vector pRS316, under the control of a strong inducible promoter GAL1 (22). This promoter is induced when cells are grown on galactose as the carbon source, and repressed on glucose. The library was introduced into the YPH275 strain, which contains a supernumerary CF that is dispensable for viability (2). The CF is telocentric, containing a long arm derived from a native yeast chromosome and functional centromere and telomere sequences (Fig. 1A). Mitotic stability of the CF is comparable to that of native yeast chromosomes (24). The CF is marked with an ochre suppressor tRNA gene SUP11, and the strain is homozygous for an ochre allele ade2-101, which results in the accumulation of a red pigment. In diploid cells, one copy of SUP11 partially suppresses the red color phenotype, making pink colonies, while two copies of the suppressor produce white colonies. The loss of the CF during colony growth is manifested by the appearance of red sectors on a pink colony background (Fig. 1B), while gain of extra copies of the CF appears as white sectors (21).
cDNA library transformants were plated on SC-Ura with galactose as the carbon source. Clones showing plasmid-dependent and galactose-dependent increases in the rate of chromosome loss or non-disjunction were selected, and the corresponding plasmids were recovered. The inserts were compared to each other using restriction mapping, Southern hybridization and partial sequencing.
Screening of approximately 105 transformants resulted in the identification of 30 different genes, which we named CST (chromosome stability). One gene (CST1/UBI2) was recovered nine times, five genes were recovered twice each and 24 genes were obtained once each.
To estimate the minimal number of CST genes that may have dosage effects on chromosome stability, we disregarded the apparently overabundant CST1 gene and assumed that the remaining genes have identical abundance in the cDNA library. An estimate based on Poisson distribution shows that at least 58 additional CST genes have not been encountered in this screen (Materials and Methods). Since different cDNAs vary in abundance, and efficiency of cDNA synthesis varies, the actual number of CST genes in yeast genome is higher than provided by this estimate.
Twenty-four CST genes correspond to previously described genes, and six are new (Table 2). Most are represented by full-length open reading frames, while some are expressed as C-terminal fragments. As expected, the known CST genes are involved in chromatin organization, cell cycle control, mitotic chromosome segregation or DNA replication. Four genes are involved in ubiquitin-directed proteolysis, which controls key steps in cell cycle progression and other processes. An unexpected finding is the identification of three components of the Ran GTPase molecular switch system. These proteins are involved in nucleocytoplasmic transport; however, no other proteins essential for this process (e.g. nucleoporins) have been obtained in this screen. This may be an indication that the Ran GTPase system also controls processes other than nucleocytoplasmic transport, as suggested previously (25).
Table 2. Identities of CST genes
| Gene | Identity | ORF (first/total amino acids) | Relevant functions or properties |
| Chromatin components or regulators | |||
| CST3 | HOS1 | Complete | Histone deacetylase |
| CST5 | RME1 | Complete | Transcription factor affecting chromatin structure |
| CST8 | NAT1 | 590/854 | N-terminal acetyltransferase, required for silencing |
| CST10 | ANC1 | Complete | Component of SNF/SWI remodeling complex |
| CST19 | HHT2 | Complete | Histone H3 |
| CST21 | RPA34 | 78/233 | Interacts with RNA polymerase I and topo I |
| CST22 | HHF2 | Complete | Histone H4 |
| CST24 | FUN30 | 550/1131 | Homolog of chromatin remodeling factor SNF2 |
| CST25 | EGD1 | Complete | Regulator of Gal4p DNA-binding activity |
| Ubiquitin-directed proteolysis | |||
| CST1 | UBI2 | Complete | Polyubiquitin precursor |
| CST2 | UBI1 | Complete | Polyubiquitin precursor |
| CST27 | PUP3 | Complete | Proteasome subunit |
| CST28 | PRE1 | Complete | Proteasome subunit |
| Cell cycle-related factors | |||
| CST7 | CLN3 | 475/580 | G1 cyclin |
| CST23 | CDC19 | Complete | Pyruvate kinase, necessary for G1/S transition |
| DNA replication | |||
| CST15 | RFC2 | Complete | Replication factor C subunit |
| CST30 | RNR2 | Complete | Ribonucleotide reductase subunit |
| Microtubule-associated proteins | |||
| CST14 | YTM1 | Complete | Microtubule-interacting protein |
| CST16 | CIN8 | 829/1038 | Kinesin-like mitotic motor |
| Nucleocytoplasmic transport (Ran GTPase system) | |||
| CST12 | RNA1 | Complete | Ran GTPase activating protein |
| CST17 | GSP1 | Complete | Ran GTPase |
| CST20 | YRB1 | Complete | Ran-binding protein 1 |
| Other | |||
| CST11 | PAB1 | Complete | Poly(A)+ RNA-binding protein |
| CST18 | SBA1 | Complete | Hsp90-associated co-chaperone |
| Novel CST genes | |||
| CST4 | YLR226W | 321/395 | Transcript peaks at mitosisa |
| CST6 | YIL036W | 207/587 | |
| CST9 | YLR394W | 268/482 | |
| CST13 | YBR158W | Complete | |
| CST26 | YBR042C | Complete | Transcript level is increased at G1/Sa |
| CST29 | YLR222C | 459/817 | |
Cellular functions affected by CST overexpression
Chromosomal instability may result from defects in cell cycle progression, DNA replication or repair or mitotic chromosome segregation. To establish which chromosomal functions are primarily affected by overexpression of the CST genes, we have determined the patterns of genomic instability, interactions with checkpoint mutations and sensitivity to hydroxyurea and benomyl, which selectively affect chromosomal functions (Table 3 and Fig. 2).
Figure 2. Examples of selective interaction of CST overexpression with growth inhibitors. Cells containing the control plasmid pRS316 or the indicated CST genes were streaked on SC, galactose-Ura plates containing HU or Benomyl (Ben.) and grown at 30°C.
Table 3. Phenotypes and checkpoint interactions produced by overexpression of selected CST genes
| Gene | Genomic instability (fold increase) | Checkpoint interactions | Drug sensitivity | |||||||
| 1:0 | 2:0 | Rec. | Mec1 | rad9 | mad1 | mad2 | mad3 | HU | Ben. | |
| Mitotic chromosome segregation phenotypes | ||||||||||
| CIN8 | w.t. | 14 | w.t. | + | + | + | - | + | + | +/- |
| CST4 | 10 | 5 | w.t. | + | + | + | + | + | + | +/- |
| DNA metabolism phenotypes | ||||||||||
| RFC2 | 10 | w.t. | 6 | - | + | + | + | + | +/- | + |
| CST6 | 12 | 4 | 6 | - | + | + | + | + | +/- | + |
| Combination phenotypes | ||||||||||
| YRB1 | 25 | 50 | 40 | - | - | - | - | +/- | - | - |
| NAT1 | 6 | w.t | 8 | - | +/- | + | + | + | - | +/- |
| CST9 | 10 | 8 | 9 | +/- | + | + | + | + | +/- | +/- |
| CST29 | 12 | 5 | 12 | +/- | + | +/- | +/- | +/- | +/- | +/- |
| Uninformative phenotypes | ||||||||||
| CST13 | 6 | 5 | 6 | + | + | + | + | + | + | + |
| CST26 | 6 | 4 | w.t. | + | + | + | + | + | + | + |
Genomic instability may result in chromosome loss (1:0 segregation) or non-disjunction (2:0), and may increase the rate of mitotic recombination. Chromosome loss is characteristic of problems in DNA metabolism, while non-disjunction reflects defects in mitotic segregation. The rate of mitotic recombination is directly related to the presence of DNA lesions (6).
Genes that cause DNA damage, replication problems or chromosome segregation defects are expected to differ from each other in the way they interact with cell cycle checkpoint mutations. The product of RAD9 gene is necessary to delay cell cycle progression in response to DNA damage, and MEC1 controls cell cycle progression in response to both DNA damage and incomplete DNA replication (26,27). Certain mutations, including mad1, mad2 and mad3, relieve the dependence of cell cycle progression on proper mitotic spindle function, allowing the cell to attempt division in the absence of chromosome segregation, which leads to cell death (28,29).
HU is an inhibitor of ribonucleotide reductase, an enzyme necessary for DNA synthesis (reviewed in 30). Benomyl is a microtubule depolymerizing drug which prevents formation of the mitotic spindle.
A specific defect in DNA metabolism should be manifested as increased 1:0 but not 2:0 segregation, elevated mitotic recombination, interaction with rad9 and/or mec1 but not mad checkpoint mutations, and sensitivity to HU but not benomyl. A defect in mitotic segregation should have the opposite effect by the above criteria.
We have applied these criteria to the CIN8 kinesin-like mitotic motor, a DNA replication factor RFC2, and to YRB1, which affects multiple nuclear functions. As expected, CIN8 fits the `mitotic' set of characteristics, in that it has increased mitotic non-disjunction (2:0), no increase in mitotic recombination, interacts with a mitotic checkpoint but not with DNA damage nor replication checkpoints, and is sensitive to benomyl but not HU (Table 3). The effects of overproduction of the novel gene CST4 are very similar, indicating that this gene is likely specifically involved in mitosis. Replication factor RFC2 fits the profile of a `DNA metabolism' gene, with increased recombination but not mitotic non-disjunction, interaction with a DNA replication checkpoint, and sensitivity to HU but not benomyl. CST6 fits the same profile, and thus may also be involved in some aspect of DNA metabolism. YRB1 has a complex pleiotropic phenotype, being positive in all assays (Table 3). This is similar to overexpression phenotypes of GSPI or RNA1, two other components of the Ran regulatory system obtained in this screen (31; data not shown). CST29 also probably affects multiple nuclear functions, since it is positive in all assays (Table 3). Phenotypes of CST13 and CST26 are not sufficiently informative to assign a specific function to these genes.
We have also determined the sensitivity of CST-overexpressing cells to MMS-induced DNA damage, and the frequency of point mutations using canavanine resistance assay. No significant effects of CST overexpression on these parameters were detected (data not shown).
CST gene deletions
We have created deletions of five genes obtained in this screen, CST9, CST13, NAT1, SBA1 and FUN30. These genes are not essential for viability. Deletions of NAT1 and SBA1 result in an increase in chromosome loss, while the other three genes have no apparent effect on chromosome stability (Table 4). Cell deleted for CST13 grow more slowly than wild type at all temperatures. Other phenotypes include temperature sensitivity of natl and fun30 deletion mutants, and HU-sensitivity of nat1 deletion.
Table 4. CST gene deletion phenotypes
| Gene | Chromosome loss | Other phenotypes |
| CST9 | No | |
| CST13 | No | Slow growth |
| NAT1 | Yes | ts-, HU sensitive |
| SBA1 | Yes | |
| FUN30 | No | ts- |
Human genes that affect chromosome stability in yeast
Since most CST genes are conserved between yeast and humans, we sought to determine whether the system based on overexpression in yeast can be used to identify human genes important for genomic stability, as suggested by (17). We used two human cDNA libraries, one in a low copy (CEN) vector under the control of the ADH promoter (32), and the other in a high copy (2 µm) vector with the GAP promoter (33).
In the screen of approximately 3 × 105 clones from the first library (CEN ADH) we identified three clones, which contained histone H2B, the C-terminal part of the small subunit of ribonucleotide reductase (the human homolog of RNR2 obtained in the yeast screen), and a mitotic cyclin B2.
The screen of approximately 4 × 105 transformants with the second library (2 µm GAP) yielded about 20 consistently sectoring clones. All of them grew slowly, with frequent appearance of revertants for both chromosome loss and slow growth. We were unable to rescue plasmids from these clones which would confer chromosome loss upon re-introduction back into the tester yeast strain. It is possible that the constitutively high level of expression from the high-copy library precludes the identification of proteins affecting chromosome. Alternatively, the difference between the results of the two screens may be related to different cDNA representation in the two libraries.
DISCUSSION
Parameters and specificity of the screen
We employed the colony color sectoring assay using a SUP11-marked CF. It was previously demonstrated that segregation of the CF is representative of behavior of native yeast chromosomes (2). Mitotic stability of the CF is very high, although slightly reduced compared to native yeast chromosomes, making the system more sensitive to conditions affecting chromosome stability. An additional advantage of this selection system is the possibility to visually distinguish between two types of chromosome transmission defects: non-disjunction (2:0 segregation) and chromosome loss (1:0), by the colony color sectoring. Since the SUP11 marker on the CF is flanked by pBR322 plasmid sequences, the marker loss generally occurs by loss of the whole CF, and not by recombination or gene conversion. This system has been successfully used to isolate a collection of mutants with reduced chromosome transmission fidelity (ctf mutants) (2). The CTF genes proved very informative for understanding the molecular bases of chromosome stability. For example, some CTF genes encode components of kinetochore, or subunits of the DNA replication complex (10,34).
Protein overexpression is a valuable tool in studying gene function. Using a high-copy yeast genomic library, two new genes that affect chromosome stability were identified (17). One of these, MIF2, is required for mitotic spindle function in anaphase, and shares sequence similarity to human kinetochore protein CENP-C (15,35). Genetic interaction between gene overexpression and temperature-sensitive mutations has proved useful in identifying specific interactions between proteins involved in kinetochore function, and between components of DNA replication machinery (20).
In the present work, we applied this approach to a yeast cDNA library under the control of strong inducible promoter, which facilitates the identification of genes with deleterious effects on cell growth and circumvents some of the limitations of high-copy libraries (discussed in 36).
Since there are numerous examples of protein overexpression producing a phenotype similar to a mutation in the corresponding gene, we expected to recover a set of genes partially overlapping that of mutations that affect chromosome segregation. This turned out to be the case. Defects in chromosome transmission have been reported for several previously described genes obtained in this screen. These include a mitotic motor CIN8, core histones HHT2 and HHF2, chromatin regulator ANC1, and the components of the Ran regulatory system, GSP1, RNA1 and YRB1. This shows that the approach based on overexpression can identify genes important for genomic stability.
The previously known genes encountered in our screen belong to functional groups related to chromatin organization, cell cycle control, microtubule function, DNA replication and proteolysis. All of these functions are necessary for proper chromosome dynamics, a requisite of genomic stability. This further demonstrates the utility of overexpression-based approach for the study of the mechanisms of chromosome stability. Interestingly, a gene homologous to poly(A)+ binding protein encoded by PAB1/CST11 and a component of ubiquitin-directed proteolysis machinery have been obtained in a similar overexpression screen in fission yeast (16).
Twenty-four out of 30 CST genes identified in this work were recovered only once each. This indicates that the screen is far from saturation, as can be expected for the relatively small number of clones screened (1.2 × 105). A minimal estimate puts the number of additional CST-type genes at 58. The spectrum of genes encountered in this screen also suggests that the screen is far from saturation. For example, given that two proteasome subunits (PUP3 and PRE1) have dosage effects on chromosome stability, it is likely that so will many others. Thus the number of CST-type genes in yeast is likely high, and the overexpression-based screening provides a convenient way to identify them.
Phenotypes produced by overexpression of CST genes
We have used several secondary criteria to identify the cellular processes affected by overexpression of CST genes: the patterns of chromosome instability, interaction with checkpoint mutations and sensitivity to agents affecting either DNA replication or mitotic spindle assembly.
Overexpression of the C-terminal portion of a kinesin-like mitotic spindle motor Cin8p produced a specifically mitotic set of traits, which may be due to its interference with the interaction between the endogenous Cin8p motor and its `cargo'. The phenotype induced by overexpression of a replication factor C subunit Rfc2p clearly points to its function in DNA replication. Overexpression of YRB1, which affects multiple nuclear functions (31), caused a combination phenotype, as expected. A similar effect was produced by the overexpression of the C-terminal part of NAT1, a subunit of the N-terminal N-acetyl transferase. This enzyme modifies and presumably regulates numerous proteins (37), and likely affects many cellular functions. Thus our criteria can distinguish genes involved in different aspects of chromosome dynamics.
When applied to novel CST genes identified in this work, the same criteria pointed to CST4 as a mitotic gene and to CST6 as a DNA metabolism gene, and to CST9 and CST29 as possible global regulators of chromosome dynamics. The mitotic phenotype of CST4 is in line with the regulation of its mRNA level, which is increased at mitosis (38). Interestingly, CST9 has sequence similarity to the RING family of DNA-binding proteins. The specific functions of these genes remain to be determined, and the information obtained from their over-expression phenotype may provide a valuable guide in this process.
Deletions of CST genes
We have created deletions of five CST genes, two of which are novel and three have been previously described, but not associated with chromosome stability functions. Deletions of NAT1 and SBA1 have a chromosome loss phenotype similar to that produced by their overexpression. In the case of NAT1, sensitivity to HU is a property of both the overexpression and deletion phenotypes. It appears that NAT1 overexpression acts by a dominant negative mechanism. Since Natlp is one of the two subunits of N-terminal N-acetyl transferase, it is likely that overexpression of the C-terminal portion of Natlp interferes with the formation of the active heterodimer.
Evolutionary conservation of CST genes
It was proposed that overexpression of heterospecific genes in yeast may be used to identify chromosome stability factors (17). We have tested this prediction for human cDNA libraries expressed in yeast, and obtained three genes similar to those recovered in the yeast cDNA screen: a core histone, a ribonuclotide reductase subunit and a cyclin. These proteins are very highly conserved in eukaryotes. The frequency of recovering human CST-type genes in yeast is ~10-fold lower than that of recovering yeast genes, which probably reflects both the greater number of genes in the human genome and the fact that the genes which are not sufficiently conserved may not have effect in a heterospecific environment. This means that screening for yeast CST genes, followed by identification of their human homologs, may be the preferential route to identification of human genes involved in chromosome stability.
Our work shows that chromosome integrity and segregation are very sensitive to the balance of components of multimolecular complexes involved in chromatin organization, ubiquitin-directed proteolysis, cell cycle control, DNA replication and mitotic spindle function. This may be one of the underlying mechanisms of genomic instability at the early stages of cancer, which are accompanied by deregulation of gene expression.
ACKNOWLEDGEMENTS
We thank P. Hieter, F. Spencer, L. Hartwell, T. Weinert, A. Murray and A. Wach for gifts of strains and plasmids, A. Bretscher for the gift of cDNA library, K. Bloom, M. Resnick, F. Spencer and V. Lundblad for discussions, members of S. Elledge laboratory for advice and DuPont Agricultural Products for the gift of benomyl. This work was supported by the American Heart Association, Texas Affiliate to I.I.O., and NIH Grants CA41424 to B.R.B. and GM44664 to S.J.E. S.J.E. is an Investigator with the Howard Hughes Medical Institute.
REFERENCES
*To whom correspondence should be addressed at: Department of Cell Biology, 126A, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Tel: +1 713 798 5295; Fax: +1 713 790 0545; Email: iliao{at}bcm.tmc.edu
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X. L. Chen, H. R. Silver, L. Xiong, I. Belichenko, C. Adegite, and E. S. Johnson Topoisomerase I-Dependent Viability Loss in Saccharomyces cerevisiae Mutants Defective in Both SUMO Conjugation and DNA Repair Genetics, September 1, 2007; 177(1): 17 - 30. [Abstract] [Full Text] [PDF]
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K. W. Y. Yuen, C. D. Warren, O. Chen, T. Kwok, P. Hieter, and F. A. Spencer Systematic genome instability screens in yeast and their potential relevance to cancer PNAS, March 6, 2007; 104(10): 3925 - 3930. [Abstract] [Full Text] [PDF]
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X. An, Z. Zhang, K. Yang, and M. Huang Cotransport of the Heterodimeric Small Subunit of the Saccharomyces cerevisiae Ribonucleotide Reductase Between the Nucleus and the Cytoplasm Genetics, May 1, 2006; 173(1): 63 - 73. [Abstract] [Full Text] [PDF]
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Z. Zhang, X. An, K. Yang, D. L. Perlstein, L. Hicks, N. Kelleher, J. Stubbe, and M. Huang Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein PNAS, January 31, 2006; 103(5): 1422 - 1427. [Abstract] [Full Text] [PDF]
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T. D. Miles, J. Jakovljevic, E. W. Horsey, P. Harnpicharnchai, L. Tang, and J. L. Woolford Jr Ytm1, Nop7, and Erb1 Form a Complex Necessary for Maturation of Yeast 66S Preribosomes Mol. Cell. Biol., December 1, 2005; 25(23): 10419 - 10432. [Abstract] [Full Text] [PDF]
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B. Montpetit, K. Thorne, I. Barrett, K. Andrews, R. Jadusingh, P. Hieter, and V. Measday Genome-Wide Synthetic Lethal Screens Identify an Interaction Between the Nuclear Envelope Protein, Apq12p, and the Kinetochore in Saccharomyces cerevisiae Genetics, October 1, 2005; 171(2): 489 - 501. [Abstract] [Full Text] [PDF]
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M. Holzel, M. Rohrmoser, M. Schlee, T. Grimm, T. Harasim, A. Malamoussi, A. Gruber-Eber, E. Kremmer, W. Hiddemann, G. W. Bornkamm, et al. Mammalian WDR12 is a novel member of the Pes1-Bop1 complex and is required for ribosome biogenesis and cell proliferation J. Cell Biol., August 1, 2005; 170(3): 367 - 378. [Abstract] [Full Text] [PDF]
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A. Geissenhoner, C. Weise, and A. E. Ehrenhofer-Murray Dependence of ORC Silencing Function on NatA-Mediated N{alpha} Acetylation in Saccharomyces cerevisiae Mol. Cell. Biol., December 1, 2004; 24(23): 10300 - 10312. [Abstract] [Full Text] [PDF]
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 K. A. Borkovich, L. A. Alex, O. Yarden, M. Freitag, G. E. Turner, N. D. Read, S. Seiler, D. Bell-Pedersen, J. Paietta, N. Plesofsky, et al. Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism Microbiol. Mol. Biol. Rev., March 1, 2004; 68(1): 1 - 108. [Abstract] [Full Text] [PDF]
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M. J. Seewald, A. Kraemer, M. Farkasovsky, C. Korner, A. Wittinghofer, and I. R. Vetter Biochemical Characterization of the Ran-RanBP1-RanGAP System: Are RanBP Proteins and the Acidic Tail of RanGAP Required for the Ran-RanGAP GTPase Reaction? Mol. Cell. Biol., November 15, 2003; 23(22): 8124 - 8136. [Abstract] [Full Text] [PDF]
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R. Yao, Z. Zhang, X. An, B. Bucci, D. L. Perlstein, J. Stubbe, and M. Huang Subcellular localization of yeast ribonucleotide reductase regulated by the DNA replication and damage checkpoint pathways PNAS, May 27, 2003; 100(11): 6628 - 6633. [Abstract] [Full Text] [PDF]
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A. A. Kitazono and S. J. Kron An Essential Function of Yeast Cyclin-dependent Kinase Cdc28 Maintains Chromosome Stability J. Biol. Chem., December 6, 2002; 277(50): 48627 - 48634. [Abstract] [Full Text] [PDF]
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S. Mehta, X. M. Yang, C. S. Chan, M. J. Dobson, M. Jayaram, and S. Velmurugan The 2 micron plasmid purloins the yeast cohesin complex: a mechanism for coupling plasmid partitioning and chromosome segregation? J. Cell Biol., August 19, 2002; 158(4): 625 - 637. [Abstract] [Full Text] [PDF]
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O. Kerscher, P. Hieter, M. Winey, and M. A. Basrai Novel Role for a Saccharomyces cerevisiae Nucleoporin, Nup170p, in Chromosome Segregation Genetics, April 1, 2001; 157(4): 1543 - 1553. [Abstract] [Full Text]
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X. Zhao, B. Georgieva, A. Chabes, V. Domkin, J. H. Ippel, J. Schleucher, S. Wijmenga, L. Thelander, and R. Rothstein Mutational and Structural Analyses of the Ribonucleotide Reductase Inhibitor Sml1 Define Its Rnr1 Interaction Domain Whose Inactivation Allows Suppression of mec1 and rad53 Lethality Mol. Cell. Biol., December 1, 2000; 20(23): 9076 - 9083. [Abstract] [Full Text]
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