Cell Cycle Arrest of Cdc Mutants and Specificity of the Rad9 Checkpoint

In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G(2) phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G(2) phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G(2) phase.     

Accumulation of Single-Stranded DNA and Destabilization of Telomeric Repeats in Yeast Mutant Strains Carrying a Deletion of RAD27

The Saccharomyces cerevisiae RAD27 gene encodes the yeast homologue of the mammalian FEN-1 nuclease, a protein that is thought to be involved in the processing of Okazaki fragments during DNA lagging-strand synthesis. One of the predicted DNA lesions occurring in rad27 strains is the presence of single-stranded DNA of the template strand for lagging-strand synthesis. We examined this prediction by analyzing the terminal DNA structures generated during telomere replication in rad27 strains. The lengths of the telomeric repeat tracts were found to be destabilized in rad27 strains, indicating that naturally occurring direct repeats are subject to tract expansions and contractions in such strains. Furthermore, abnormally high levels of single-stranded DNA of the templating strand for lagging-strand synthesis were observed in rad27 cells. Overexpression of Dna2p in wild-type cells also yielded single-stranded DNA regions on telomeric DNA and caused a cell growth arrest phenotype virtually identical to that seen for rad27 cells grown at the restrictive temperature. Furthermore, overexpression of the yeast exonuclease Exo1p alleviated the growth arrest induced by both conditions, overexpression of Dna2p and incubation of rad27 cells at 37 degrees C. However, the telomere heterogeneity and the appearance of single-stranded DNA are not prevented by the overexpression of Exo1p in these strains, suggesting that this nuclease is not simply redundant with Rad27p. Our data thus provide in vivo evidence for the types of DNA lesions predicted to occur when lagging-strand synthesis is deficient and suggest that Dna2p and Rad27p collaborate in the processing of Okazaki fragments.     

Stage-Specific Effects of X-Irradiation on Yeast Meiosis

Previous work has shown that cdc13 causes meiotic arrest of Saccharomyces cerevisiae following DNA replication by a RAD9-dependent mechanism. In the present work, we have further investigated the implicit effects of chromosomal lesions on progression through meiosis by exposing yeast cells to X-irradiation at various times during sporulation. We find that exposure of RAD9 cells to X-irradiation early in meiosis prevents sporulation, arresting the cells at a stage prior to premeiotic DNA replication. rad9 meiotic cells are much less responsive to X-irradiation damage, completing sporulation after treatment with doses sufficient to cause arrest of RAD9 strains. These findings thereby reveal a RAD9-dependent checkpoint function in meiosis that is distinct from the G(2) arrest previously shown to result from cdc13 dysfunction. Analysis of the spores that continued to be produced by either RAD9 or rad9 cultures that were X-irradiated in later stages of sporulation revealed most spores to be viable, even after exposure to radiation doses sufficient to kill most vegetative cells. This finding demonstrates that the lesions induced by X-irradiation at later times fail to trigger the checkpoint function revealed by cdc13 arrest and suggests that the lesions may be subject to repair by serving as intermediates in the recombination process. Strains mutant for chromosomal synapsis and recombination, and therefore defective in meiotic disjunction, were tested for evidence that X-ray-induced lesions might alleviate inviability by promoting recombination. Enhancement of spore viability when spo11 (but not hop1) diploids were X-irradiated during meiosis indicates that induced lesions may partially substitute for SPO11-dependent functions that are required for the initiation of recombination.     

Characterization of G(1) Checkpoint Control in the Yeast Saccharomyces Cerevisiae following Exposure to DNA-Damaging Agents

The delay of S-phase following treatment of yeast cells with DNA-damaging agents is an actively regulated response that requires functional RAD9 and RAD24 genes. An analysis of cell cycle arrest indicates the existence of (at least) two checkpoints for damaged DNA prior to S-phase; one at START (a G(1) checkpoint characterized by pheromone sensitivity of arrested cells) and one between the CDC4- and CDC7-mediated steps (termed the G(1)/S checkpoint). When a dna1-1 mutant (that affects early events of replicon initiation) also carries a rad9 deletion mutation, it manifests a failure to arrest in G(1)/S following incubation at the restrictive temperature. This failure to execute regulated G(1)/S arrest is correlated with enhanced thermosensitivity of colony-forming ability. In an attempt to characterize the signal for RAD9 gene-dependent G(1) and G(1)/S cell cycle arrest, we examined the influence of the continued presence of unexcised photoproducts. In mutants defective in nucleotide excision repair, cessation of S-phase was observed at much lower doses of UV radiation compared to excision-proficient cells. However, this response was not RAD9-dependent. We suggest that an intermediate of nucleotide excision repair, such as DNA strand breaks or single-stranded DNA tracts, is required to activate RAD9-dependent G(1) and G(1)/S checkpoint controls.     

Cdc20, a β-transducin homologue,links RAD9-mediated G2/M checkpoint control to mitosis in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the DNA damage-induced G2 arrest requires the checkpoint control genes RAD9, RAD17, RAD24, MEC1, MEC2 and MEC3. These genes also prevent entry into mitosis of a temperature-sensitive mutant, cdc13, that accumulates chromosome damage at 37^°?C. Here we show that a cdc13 mutant overexpressing Cdc20, a β-transducin homologue, no longer arrests in G2 at the restrictive temperature but instead undergoes nuclear division, exits mitosis and enters a subsequent division cycle, which suggests that the DNA damage-induced G2/M checkpoint control is not functional in these cells. This is consistent with our observation that overexpression of CDC20 in wild-type cells results in increased sensitivity to UV irradiation. Overproduction of Cdc20 does not influence the arrest phenotype of the cdc mutants whose cell cycle block is independent of RAD9-mediated checkpoint control. Therefore, we suggest that the DNA damage-induced checkpoint controls prevent mitosis by inhibiting the nuclear division pathway requiring CDC20 function.     

The Saccharomyces recombination protein Tid1p is required for adaptation from G2/M arrest induced by a double-strand break

Saccharomyces cells with a single unrepaired double-strand break (DSB) will adapt to checkpoint-mediated G2/M arrest and resume cell cycle progression. The decision to adapt is finely regulated by the extent of single-stranded DNA generated from a DSB. We show that cells lacking the recombination protein Tid1p are unable to adapt, but that this defect is distinct from any role in recombination. As with the adaptation-defective mutations yku70Delta and cdc5-ad, permanent arrest in tid1Delta is bypassed by the deletion of the checkpoint gene RAD9. Permanent arrest of tid1Delta cells is suppressed by the rfa1-t11 mutation in the ssDNA binding complex RPA, similar to yku70Delta, whereas the defect in cdc5-ad is not suppressed. Unlike yku70Delta, tid1Delta does not affect 5'-to-3' degradation of DSB ends. The tid1Delta defect cannot be complemented by overexpressing the homolog Rad54p, nor is it affected in rad51Delta tid1Delta, rad54Delta tid1Delta, or rad52Delta tid1Delta double mutants that prevent essentially all homologous recombination. We suggest that Tid1p participates in monitoring the extent of single-stranded DNA produced by resection of DNA ends in a fashion that is distinct from its role in recombination.     

Rad24 truncation,coupled with altered telomere structure,promotes cdc13-1 suppression in S. cerevisiae

Distinguishing telomeres from DNA double strand breaks is critical for genome stability. In S. cerevisiae, the Cdc13 single-strand telomere binding protein is critical for protecting chromosome ends. The C-rich telomere strand is lost at high temperatures in cdc13-1 strains, leading to activation of the DNA damage checkpoint and cell inviability. Through a screen performed to identify activities involved in telomere C-strand loss, we identified two new rad24 alleles. Rad24 is an alternate Rfc1 subunit, functioning to load the 9-1-1 checkpoint clamp. In each rad24 allele, a transposon inserted within the RAD24 coding region leads to expression of different carboxyl-terminal portions of Rad24, deleting or truncating the amino-terminus. We show that an intact Rad24 amino-terminus is necessary for its checkpoint function. Interestingly, the initial cdc13-1 rad24-2 strains grew at 36Ã?Â?Å¡C, but the extent of suppression associated with rad24-2 weakened in serial backcrosses, and cdc13-1 segregants from these crosses showed a modest increase in temperature resistance. Moreover, while a RAD24 plasmid suppressed the checkpoint defect in the initial cdc13-1 rad24-2 strain, the temperature resistance was only partially suppressed. These data suggest that the TG1-3 amplification observed in this strain contributes to the suppression phenotype. By recreating the rad24-2 allele in a strain with normal telomeres, we find that, relative to the rad24-Ã?¢Ã?Â?†allele, rad24-2 increases the frequency of obtaining cdc13-1 cells capable of growth at high temperatures. Our hypothesis is that the Rad24-2 truncation protein affects telomere structure or recombination in a manner distinct from rad24-Ã?¢Ã?Â?†.     

The DNA damage checkpoint allows recombination between divergent DNA sequences in budding yeast

In the early steps of homologous recombination, single-stranded DNA (ssDNA) from a broken chromosome invades homologous sequence located in a sister or homolog donor. In genomes that contain numerous repetitive DNA elements or gene paralogs, recombination can potentially occur between non-allelic/divergent (homeologous) sequences that share sequence identity. Such recombination events can lead to lethal chromosomal deletions or rearrangements. However, homeologous recombination events can be suppressed through rejection mechanisms that involve recognition of DNA mismatches in heteroduplex DNA by mismatch repair factors, followed by active unwinding of the heteroduplex DNA by helicases. Because factors required for heteroduplex rejection are hypothesized to be targets and/or effectors of the DNA damage response (DDR), a cell cycle control mechanism that ensures timely and efficient repair, we tested whether the DDR, and more specifically, the RAD9 gene, had a role in regulating rejection. We performed these studies using a DNA repair assay that measures repair by single-strand annealing (SSA) of a double-strand break (DSB) using homeologous DNA templates. We found that repair of homeologous DNA sequences, but not identical sequences, induced a RAD9-dependent cell cycle delay in the G2 stage of the cell cycle. Repair through a divergent DNA template occurred more frequently in RAD9 compared to rad9Δ strains. However, repair in rad9Δ mutants could be restored to wild-type levels if a G2 delay was induced by nocodazole. These results suggest that cell cycle arrest induced by the Rad9-dependent DDR allows repair between divergent DNA sequences despite the potential for creating deleterious genome rearrangements, and illustrates the importance of additional cellular mechanisms that act to suppress recombination between divergent DNA sequences.     

A Rad9-Dependent Checkpoint Blocks Meiosis of Cdc13 Yeast Cells

Mutations in CDC13 have previously been found to cause cell cycle arrest of Saccharomyces cerevisiae at a stage in G2 immediately preceding the mitotic division. We show here that cdc13 blocks the meiotic pathway at a stage that follows DNA replication, but in this case the spindle has not yet formed nor have the chromosomes undergone synapsis or recombination. This arrest is alleviated by rad9, thus implicating the same checkpoint function that delays mitotic progression when chromosomal lesions are present. An assessment of the spores produced upon alleviation of the meiotic arrest by rad9 reveals that the absence of recombination in strains bearing cdc13 alone is attributable to the RAD9-mediated arrest rather than to other effects of cdc13 lesions. We have tested the possibility that this checkpoint function is important in regulating meiotic progression to permit resolution of recombinational intermediates during ongoing meiosis and have found no evidence that rad9 alters the execution of functions that might depend upon such regulation. We consider the possible role of other checkpoints in yeast meiosis.     

Incision and postincision steps of pyrimidine dimer removal in excision-defective mutants of Saccharomyces cerevisiae.

cdc9, a temperature-sensitive mutant defective in polynucleotide deoxyribonucleic acid (DNA) ligase activity, accumulates low-molecular-weight DNA fragments (as measured by sedimentation of DNA in alkaline sucrose gradients) at the nonpermissive temperature after irradiation with ultraviolet light. This phenotype of cdc9 is a sensitive indicator of successful incision during excision repair of dimers. In strains containing excision-defective mutations in any of nine genes in combination with the cdc9 mutation, the absence of low-molecular-weight DNA at the nonpermissive temperature after ultraviolet treatment suggests that these mutants are incision defective, whereas the presence of low-molecular-weight DNA indicates that the mutants are defective in a step after incision. With rad1, rad2, rad3, rad4, and rad10 mutants, the molecular weight of the DNA remained unchanged after ultraviolet irradiation and incubation at the restrictive temperature, despite the presence of the cdc9 mutation; these mutants are therefore incision defective. Low-molecular-weight DNA was observed in rad14 cdc9 and rad16 cdc9 strains. With the rad16 strain, the accumulation of low-molecular-weight DNA correlated with the amount of excision taking place, whereas in the rad14 mutant strain, no evidence of dimer removal was obtained. Therefore, rad14 is likely to be defective in a step after incision.     

DNA-repair characterization of cdc40-1, a cell-cycle mutant of Saccharomyces cerevisiae

The cell-cycle specific mutation cdc40-1, which has been previously shown to be sensitive to MMS at the restrictive temperature, was further characterized as a DNA-repair-deficient mutation. cdc40-1 mutants shown only slight sensitivity to UV irradiation. Double mutant studies shown that rad6-l is epistatic to cdc40-1 with respect to sensitivity to UV irradiation and MMS. rad50-1 is epistatic to cdc40-1 with respect to MMS sensitivity in G1 stationary cells, but not in logarithmic cultures. An additive effect is seen between cdc40-1 and rad50-1 with respect to UV irradiation. cdc40-1 mutants are defective in UV-induced mutagenesis at the restrictive temperature. UV-induced levels of recombination are normal at both temperatures, while MMS-induced recombination is enhanced at the restrictive temperature.     

Sensor and effector kinases in DNA damage checkpoint regulate capacity for homologous recombination repair of fission yeast in G2 phase

Although the G2/M DNA damage checkpoint is currently viewed as a set of coordinated cellular responses affecting both cell cycle progression and non-cell cycle targets, the relative contributions of the two target categories to DNA repair and cell survival after exposure to ionizing radiation have not been clearly addressed. We investigated how rad3 (ATR ortholog) or chk1/cds1 (CHK1/CHK2 orthologs) null mutations change the kinetics of double-strand break (DSB) repair in Schizosaccharomyces pombe cells under conditions of forced G2 arrest. After 200-Gy γ-ray irradiation, DSBs were repaired in rad3Δ cdc25-22 or chk1Δ cds1Δ cdc25-22 cells, almost as efficiently as in cdc25-22 cells at the restrictive temperature. In contrast, little repair was observed in the checkpoint-deficient cells up to 4h after higher-dose (500Gy) irradiation, whereas repair was still efficient in the control cdc25-22 cells. Immediate loss of viability appeared not be responsible for the repair defect after the higher dose, since both checkpoint-proficient and deficient cells with cdc25-22 allele synchronously resumed cycling with a similar time course when released to the permissive temperature 4h after irradiation. Recruitment of repair proteins Rad11 (Rpa1 ortholog), Rad22 (Rad52 ortholog), and Rhp54 (Rad54 ortholog) to the damage sites was not significantly impaired in the checkpoint-deficient cells, whereas their release was profoundly delayed. Our results suggest that sensor and effector kinases in the damage checkpoint machinery affect the efficiency of repair downstream of, or in parallel with the core repair reaction.     

Exposure of single-stranded telomeric DNA causes G2/M cell cycle arrest in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Cdc13p is a single-stranded TG(1-3) DNA binding protein that protects telomeres and maintains telomere length. A mutant allele of CDC13, cdc13-1, causes accumulation of single-stranded TG(1-3) DNA near telomeres along with a G(2)/M cell cycle arrest at non-permissive temperatures. We report here that when the single-stranded TG(1-3) DNA is masked by its binding proteins, such as S. cerevisiae Gbp2p or Schizosaccharomyces pombe Tcg1, the growth arrest phenotype of cdc13-1 is rescued. Mutations on Gbp2p that disrupt its binding to the single-stranded TG(1-3) DNA render the protein unable to complement the defects of cdc13-1. These results indicate that the presence of a single-stranded TG(1-3) tail in cdc13-1 cells serves as the signal for the cell cycle checkpoint. Moreover, the binding activity of Gbp2p to single-stranded TG(1-3) DNA appears to be associated with its ability to restore the telomere-lengthening phenotype in cdc13-1 cells. These results indicate that Gbp2p is involved in modulating telomere length.     

Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13

In Saccharomyces cerevisiae, Cdc13 has been proposed to mediate telomerase recruitment at telomere ends. Stn1, which associates with Cdc13 by the two-hybrid interaction, has been implicated in telomere maintenance. Ten1, a previously uncharacterized protein, was found to associate physically with both Stn1 and Cdc13. A binding defect between Stn1-13 and Ten1 was responsible for the long telomere phenotype of stn1-13 mutant cells. Moreover, rescue of the cdc13-1 mutation by STN1 was much improved when TEN1 was simultaneously overexpressed. Several ten1 mutations were found to confer telomerase-dependent telomere lengthening. Other, temperature-sensitive, mutants of TEN1 arrested at G(2)/M via activation of the Rad9-dependent DNA damage checkpoint. These ten1 mutant cells were found to accumulate single-stranded DNA in telomeric regions of the chromosomes. We propose that Ten1 is required to regulate telomere length, as well as to prevent lethal damage to telomeric DNA.