UV Irradiation Causes the Loss of Viable Mitotic Recombinants in Schizosaccharomyces pombe Cells Lacking the G₂/M DNA Damage Checkpoint

Corresponding author: Fekret Osman, University College London, Gower St., London WC1E 6BT, United Kingdom., fekret.osman{at}bioch.ox.ac.uk (E-mail)

Communicating editor: P. RUSSELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Elevated mitotic recombination and cell cycle delays are two of the cellular responses to UV-induced DNA damage. Cell cycle delays in response to DNA damage are mediated via checkpoint proteins. Two distinct DNA damage checkpoints have been characterized in Schizosaccharomyces pombe: an intra-S-phase checkpoint slows replication and a G₂/M checkpoint stops cells passing from G₂ into mitosis. In this study we have sought to determine whether UV damage-induced mitotic intrachromosomal recombination relies on damage-induced cell cycle delays. The spontaneous and UV-induced recombination phenotypes were determined for checkpoint mutants lacking the intra-S and/or the G₂/M checkpoint. Spontaneous mitotic recombinants are thought to arise due to endogenous DNA damage and/or intrinsic stalling of replication forks. Cells lacking only the intra-S checkpoint exhibited no UV-induced increase in the frequency of recombinants above spontaneous levels. Mutants lacking the G₂/M checkpoint exhibited a novel phenotype; following UV irradiation the recombinant frequency fell below the frequency of spontaneous recombinants. This implies that, as well as UV-induced recombinants, spontaneous recombinants are also lost in G₂/M mutants after UV irradiation. Therefore, as well as lack of time for DNA repair, loss of spontaneous and damage-induced recombinants also contributes to cell death in UV-irradiated G₂/M checkpoint mutants.

DAMAGE to DNA, from both endogenous and exogenous sources, is intrinsic to life. To counteract the deleterious effects of such damage, cells are equipped with an intricate network of repair pathways, including recombinational repair between homologous DNA sequences. Repair is aided by delays in the cell division cycle in response to DNA damage, providing time and opportunities to remove or tolerate DNA lesions. Loss of either of these coordinated responses to DNA damage, DNA repair and cell cycle delay, can lead to loss of DNA integrity and cell death and in mammalian cells can predispose organisms to cancer.

Cell cycle delays in response to DNA damage are imposed by checkpoint pathways, which sense DNA damage and transduce the signal to the cell cycle machinery. Two distinct checkpoints in response to DNA damage have been characterized in the fission yeast Schizosaccharomyces pombe (reviewed in CARR 1998 ; RHIND and RUSSELL 1998A ; CASPARI and CARR 1999 ; HUMPHREY 2000 ; O'CONNELL et al. 2000 ): an intra-S-phase checkpoint slows replication and a G₂/M checkpoint stops cells passing from G₂ into mitosis. The G₂/M transition is the major control point in the S. pombe cell cycle. A G₁/S DNA damage checkpoint, which prevents replication of damaged chromosomes, has not been rigorously analyzed in S. pombe and is experimentally difficult to assess. However, limited studies (OSMAN and MCCREADY 1998 ; RHIND and RUSSELL 1998B ) suggest that UV damage, but not ionizing radiation damage, activates this checkpoint in S. pombe, although it is unclear whether it is mechanistically distinct from the intra-S checkpoint.

In this study we have analyzed two functional classes of checkpoint mutants: the so-called "rad" checkpoint mutants, rad1, rad3, rad9, rad17, rad26, and hus1, disrupt an early stage of the DNA damage checkpoint and "transduction" checkpoint mutants rhp9/crb2, chk1/rad27, and cds1 are disrupted for a function downstream of the rad checkpoint proteins, but upstream of the cell cycle machinery. The rad checkpoint mutants all lack the G₂/M DNA damage checkpoint, and at least rad3 and rad26 cells also lack the intra-S-phase checkpoint. Although untested, the other rad checkpoint mutants are also likely to lack the intra-S-phase checkpoint since they all have very similar phenotypes. Of the transduction checkpoint mutants, rhp9 and chk1 lack the G₂/M checkpoint whereas cds1 mutants lack the intra-S-phase checkpoint. The intra-S-phase checkpoint remains intact in chk1 cells (and most probably in rhp9 mutants as well since they are phenotypically very similar). Thus, in broad terms, there are three main phenotypic classes of DNA damage checkpoint mutant: one that lacks the G₂/M and intra-S checkpoint (the rad checkpoint mutants), one that lacks only the G₂ checkpoint (chk1 and rhp9), and a third that lacks only the intra-S-phase checkpoint (cds1). S. pombe Rqh1, a member of the RecQ subfamily of DNA helicases, is not a checkpoint protein, but appears to play a role in an S phase arrest survival/recovery mechanism in response to UV damage (MURRAY et al. 1997 ; STEWART et al. 1997 ) that also involves Cds1 and the Rad checkpoint proteins (AL-KHODAIRY et al. 1994 ; LINDSAY et al. 1998 ).

As well as cell cycle delays, DNA damage caused by UV irradiation also stimulates mitotic homologous recombination in a variety of organisms (GROSSENBACHER- GRUNDER and THURIAUX 1981 ; FRIEDBERG et al. 1995 ; DOE et al. 2000 ; OSMAN et al. 2000 ). This indicates that recombinational pathways play a role in the repair or tolerance of UV-induced DNA damage. In S. pombe this idea is supported by the pronounced UV sensitivity of recombination mutants, such as rad22, rhp51, and rhp54 (OSTERMANN et al. 1993 ; MURIS et al. 1993 , MURIS et al. 1996 ), and by the observation that these mutants lack UV-induced mitotic recombination (OSMAN et al. 2000 ). Elevated mitotic recombination could be due to activation of the cellular recombination machinery since key recombination genes in both S. pombe (JANG et al. 1996 ) and the budding yeast Saccharomyces cerevisiae (COLE et al. 1987 ) are induced by DNA damage. Alternatively, UV lesions in DNA, either directly or following processing by excision repair enzymes, may act as initiation sites for recombination. UV-stimulated recombination could also result as a consequence of replication forks stalling at bulky DNA lesions. It is becoming increasingly apparent that recombination can rescue stalled and/or collapsed replication forks (reviewed in KUZMINOV 2001 ). Stalled forks potentially provide single-strand gaps onto which recombinases may load. They can also regress to form Holliday junctions that can be cleaved, resulting in the collapse of the replication fork and generation of a recombinogenic double-stranded end.

Recent studies have revealed unexpected links between checkpoints, recombinational repair pathways, and several human cancer predisposition and genome instability syndromes (reviewed in VAN GENT et al. 2001 ). For example, the human genetic disorder ataxia telangiectasia is caused by mutation in the ATM gene (SAVITSKY et al. 1995 ), which plays a major role in DNA damage checkpoints in human cells (reviewed in BROWN et al. 1999 ). Recent evidence suggests that ATM defects impair DNA repair mediated by homologous recombination and may link cell cycle checkpoints to the activation of homologous recombination (CHEN et al. 1999 ; MORRISON et al. 2000 ). Studies of the human p53 gene also reveal a direct relationship between cell cycle checkpoints, homologous recombination, and cancer (WEINERT and LYDALL 1993 ; STURZBECHER et al. 1996 ).

A direct link between homologous recombination and DNA damage checkpoints has also been shown in S. cerevisiae by demonstrating that the recombinational repair protein Rad55 was phosphorylated in response to DNA damage in a DNA damage checkpoint-dependent manner (BASHKIROV et al. 2000 ). This is also consistent with a role for DNA damage checkpoints in activating recombinational repair. In addition, srs2 mutants have a simultaneous hyper-recombination phenotype (ABOUSSEKHRA et al. 1992 ) and a checkpoint defect (LIBERI et al. 2000 ). Dun1 mutants are partially defective in DNA damage-induced G₂ arrest (PATI et al. 1997 ) and have enhanced levels of spontaneous and UV-induced mitotic recombination (FASULLO et al. 1999 ).

An intimate relationship between checkpoints and recombination is also illustrated by the checkpoint-dependent delay in the cell cycle exhibited by S. pombe recombination mutants such as rhp54 and rad22 (MURIS et al. 1996 ; SUTO et al. 1999 ). S. pombe survival during and recovery from damage-induced intra-S checkpoint arrest may rely on homologous recombinational pathways that either allow replication fork bypass of DNA lesions or restore stalled/collapsed replication forks (MURRAY et al. 1997 ; STEWART et al. 1997 ; DOE et al. 2000 ). Recently, it has been shown that the damage-induced transcription of the recombination gene rhp51⁺ is dependent on checkpoint proteins (SHIM et al. 2000 ).

In this study we have sought to determine whether UV damage-induced mitotic intrachromosomal recombination relies on damage-induced cell cycle delays. The spontaneous and UV-induced recombination phenotypes were determined for checkpoint mutants lacking the intra-S and/or the G₂/M checkpoint. We present evidence that suggests that the checkpoint-mediated G₂/M delay in response to UV damage is essential to allow viable recombination, both UV induced and that occurring spontaneously due to endogenous DNA damage or intrinsic stalling of replication forks.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media and genetic and molecular techniques:
Media and general genetic methods for S. pombe have been described (GUTZ et al. 1974 ). The complete medium was yeast extract medium supplemented with 225 mg/liter adenine, histidine, leucine, lysine, and uracil (YES). The minimal medium was Edinburgh minimal medium (EMM) supplemented with appropriate amino acids. The medium used to select Ade⁺ recombinants in plating assays was YES lacking adenine and supplemented with 200 mg/liter guanine to prevent adenine uptake. UV irradiation was performed using a 254-nm UV germicidal lamp.

Strains:
Genotypes of the strains used in this study are shown in Table 1. Strains were constructed by random spore analysis, where appropriate, or by tetrad dissection following a sexual cross. The ade6^- heteroallelic duplication, the intrachromosomal recombination substrate, was originally constructed in strain FO163 as described previously (OSMAN et al. 2000 ). Other ade6^- duplication strains were constructed by sexual crosses.

Standard recombination assays:
Mitotic recombination was assayed by the recovery of Ade⁺ recombinants from strains containing the intrachromosomal recombination substrate shown in Fig 1. It consists of nontandem direct repeats of ade6^- heteroalleles (ade6-L469 and ade6-M375) separated by pUC sequence and a functional his3⁺ gene (the endogenous his3⁺ gene is deleted). Frequencies of spontaneous and UV-induced recombinants were determined by fluctuation tests. For each strain, five independent colonies were used per assay. Each assay was repeated independently at least three times. Thus, for each strain at least 15 colonies were assayed. For each assay, single colonies grown for 4 days on YES complete media were used. Cells from each single colony were resuspended in sterile water and plated at a density of 10⁵–10⁶ cells per plate (10⁴–10⁵ cells per plate for the hyper-recombinant rqh1 strain) onto several plates containing media selective for Ade⁺. One of the plates was used to select spontaneous Ade⁺ recombinants. The remaining plates were irradiated with appropriate doses of UV light to select recombinant colonies arising after UV irradiation. To determine cell titers, appropriately diluted cells were spread onto several plates containing YES complete media. One of the plates was unirradiated and used to determine the unirradiated cell titer. The remaining plates were irradiated with appropriate doses of UV and used to determine the UV-irradiated cell titer. After 4 days of growth at 30° the number of recombinants and cell titer were determined. The Ade⁺ recombinant colonies on the selective plates were replicated onto EMM lacking adenine and histidine (and onto media lacking just adenine as a control) to determine the proportion of conversion-type (Ade⁺ His⁺) and deletion-type (Ade⁺ His^-) recombinants.

View larger version (28K): In this window In a new window Download PPT slide   Figure 1. Schematic of the mitotic intrachromosomal recombination substrate and two classes of Ade+ recombinants. Circles represent the ade6-L469 and ade6-M375 point mutations. The ade6- heteroalleles are nontandem direct repeats. Some of the postulated pathways that can give rise to deletion- and conversion-type recombinants are indicated.

For each strain, mean recombinant frequencies were determined for each of the three independent assays. The average recombinant frequencies and standard deviations were determined from these three means, according to LURIA and DELBRUCK 1943 . The percentage of conversion types and standard deviations were also determined in the same way. To analyze the statistical significance of differences in recombinant frequencies (and percentage of conversion types) for a given strain compared to wild-type cells, two sample t-tests were used to analyze individual recombinant frequencies (and percentage of conversion types) from all colonies (at least 15 per strain). To analyze the statistical significance of the effects of UV on recombinant frequencies, paired t-tests were used comparing paired spontaneous and induced frequencies for all individual colonies assayed.

Recombination assays on G₁ cells:
Spontaneous and UV-induced recombinant frequencies of G₁ cells were determined as described above (five independent colonies per assay, three assays per strain), except that the cells were initially synchronized in G₁ by nitrogen starvation as described previously (LINDSAY et al. 1998 ). Briefly, cells from a colony grown for 3 days on YES were incubated overnight in 5 ml of EMM at 25°. Cells were collected by centrifugation, washed three times in water, resuspended in 5 ml EMM lacking a nitrogen source and incubated for 20 hr at 25° to synchronize cells in G₁. Cells were then collected by centrifugation, washed and resuspended, and incubated in YES media to allow recovery. At this point most cells were in G₁ with a 1C DNA content (confirmed by FACS analysis, not shown). The G₁ cells were then washed again in water and appropriate dilutions were used for the recombination assays. Cells were irradiated immediately after plating to ensure that they were still in G₁.

Recombination assays using the cdc25-22 mutation to impose an artificial G₂ delay after UV irradiation:
These recombination assays using the cdc25-22 mutation to synchronize and maintain cells in G₂ (RUSSELL and NURSE 1986 ) were performed as before except for the following modifications. Cells from a colony grown at 25° for 4 days on YES were incubated in 1 ml of YES medium at 36.5° for 2.5 hr. Mitotic index and FACS analyses (not shown) confirmed that cdc25-22 cells treated in this way were synchronized in G₂ and could subsequently be maintained in G₂ at 36.5° or could be allowed to rapidly reenter the cell cycle and proceed to mitosis by changing their incubation temperature to 25°. Maintaining their temperature at 36.5°, cells were plated onto two sets of plates for each assay (plates had initially been preincubated at 36.5°). Plates were UV irradiated, where appropriate, at 36.5°. After UV irradiation, one set of plates was maintained at 36.5° to keep cells in G₂ for a further 2 hr prior to incubation at 25°. The other set of plates was immediately moved to 25° after UV irradiation, allowing cells to rapidly resume cycling.

In control experiments, temperature (25°, 30°, or 36.5°) was shown to have no effect on recombination in cdc25⁺ cells (data not shown).

UV sensitivities of G₁ cells compared to unsynchronized (mostly G₂) cells:
For each strain tested, three independent cell cultures were used to obtain UV survival curves. Cells from a cell culture were split in two. Half of the cells were synchronized in G₁ by nitrogen starvation, as described above, and the other half were allowed to continue cycling. The cells allowed to continue cycling were unsynchronized. Unsynchronized S. pombe cells mostly have a 2n DNA content and 70–80% are in G₂, which is the longest phase of their cell cycle. Cells were plated at appropriate dilutions onto YES medium (three plates per dose) and irradiated at various UV doses. Plates were incubated for 4 days and the colonies were counted.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Effect of checkpoint mutations on spontaneous recombination:
Strains containing a nontandem direct repeat of ade6^- heteroalleles (Fig 1) can be used to assay intrachromosomal mitotic recombination by determining the frequency at which Ade⁺ recombinants are recovered (DOE et al. 2000 ; OSMAN et al. 2000 ). Two main classes of Ade⁺ recombinants could be distinguished depending on whether a functional his3⁺ gene between the repeats was retained following recombination (Fig 1): Ade⁺ His^- deletion-type recombinants, in which one copy of the duplicated sequence and the intervening sequence were lost, and Ade⁺ His⁺ conversion types, which retained the duplication. Intrachromosomal direct repeats can recombine with each other to generate a recombinant product by multiple, and sometimes overlapping, pathways, as listed in Fig 1 (reviewed in KLEIN 1995 ; HABER 1995 ; OSMAN and SUBRAMANI 1998 ; PAQUES and HABER 1999 ). In S phase and G₂ cells following DNA replication, recombination can occur between sister chromatids (interchromatid) as well as within a single chromatid (intrachromatid).

Checkpoint mutant strains containing this recombination substrate were constructed and used to determine the effect of loss of checkpoint genes on recombinant frequencies. The spontaneous recombinant frequencies for wild type and checkpoint mutant cells are shown in Table 2. In wild-type cells, spontaneous Ade⁺ recombinants arose at an average frequency of 3.71 x 10^-4 per viable cell. About a third of these recombinants were conversion types and the remainder deletion types. These observations are consistent with our previous studies (DOE et al. 2000 ; OSMAN et al. 2000 ). Compared to wild-type cells, the checkpoint mutants displayed either no effect or a moderate effect on spontaneous recombinant frequencies. There were no significant differences between the frequencies of Ade⁺ recombinants obtained from wild-type cells and from rad1, rad3, rad9, rad17, rhp9, and chk1 mutant cells. These mutants also had no effect on the distribution of deletion- and conversion-type recombinants compared to wild type. The hus1 mutant was the only one with a hypo-recombinant phenotype. It exhibited a moderate (less than twofold) but significant decrease in the frequency of Ade⁺ recombinants compared to wild-type cells, but there was no significant effect on the relative distribution of deletion- to conversion-type recombinants. Mutant rad26 cells exhibited a small (less than twofold) but significant hyper-recombination phenotype compared to wild type, but had no significant effect on the distribution of recombinants.

Cds1 and chk1 cds1 cells exhibited a moderate (less than threefold) but more marked hyper-recombination phenotype with no significant effect on the distribution of recombinants. In two-sample t-tests, there were no significant differences (P > 0.05) between the frequencies of recombinants obtained from cds1 and chk1 cds1 cells. As shown previously (DOE et al. 2000 ), rqh1 cells also displayed a spontaneous hyper-recombination phenotype compared to wild-type cells (more than threefold increase in recombinants). The distribution of deletion- and conversion-type recombinants was also affected so that most extra recombinants were deletion types with conversion types accounting only for <20% of recombinants, compared to >30% for wild-type cells.

Effect of UV irradiation on recombinant frequencies in DNA damage checkpoint mutants:
Strains containing the intrachromosomal recombination substrate were used to determine the effect of loss of DNA damage checkpoints on recombinant frequencies following UV irradiation. The effect of UV on mitotic recombinant frequencies for wild-type and checkpoint mutants is shown in Fig 2. As shown previously (OSMAN et al. 2000 ), for wild-type cells the total Ade⁺ recombinant frequency was significantly elevated (P < 0.05 at all doses in paired two-sample t-tests) with increasing killing by UV (Fig 2A). UV irradiation induced both types of recombinants; however, the increase was differential and a rising proportion of the extra recombinants were conversion types (Fig 2B). Whereas deletion types outnumbered conversion types for spontaneous recombination, the reverse was true after UV irradiation giving 50% survival or less.

View larger version (44K): In this window In a new window Download PPT slide   Figure 2. Effect of UV irradiation on the frequency and distribution of recombinants in wild-type and mutant cells. (A) Effect of UV on total Ade+ recombinant frequencies. The UV dose (J/m2) used for the different strains was as follows: 0, 40, 80, 120, and 160 for wild type and cds1; 0, 10, and 20 for rad1, rad3, rad9, rad17, and rad26; 0, 5, 10, and 20 for hus1; 0, 5, 10, 20, and 30 for rhp9; 0, 30, and 60 for chk1; 0, 10, 20, and 30 for chk1 cds1; 0, 10, 20, and 40 for rqh1. (B) Effect of UV irradiation on the relative frequencies of deletion- and conversion-type recombinants for wild-type, chk1, cds1, and rqh1 cells.

Rad1, rad3, rad9, rad17, rad26, hus1, rhp9, chk1, and chk1 cds1 cells all exhibited a similar, novel phenotype: following UV irradiation, there was a sharp fall in the recombinant frequency to below the frequency of spontaneous recombinants. The decrease in recombinant frequencies correlated with increased killing by UV and was significantly (P < 0.05) reduced compared to spontaneous levels in paired two-sample t-tests. For all these mutants, there was a fall in the frequency of both deletion and conversion types with no alteration in their relative frequency, as shown for chk1 in Fig 2B (data not shown for others). Although the rad checkpoint mutants and chk1 cds1 cells lack both the intra-S and G₂/M DNA damage checkpoint, the loss of viable recombinants following UV irradiation appears to be associated with the loss of the G₂/M checkpoint since chk1 and rhp9 cells lack only the G₂/M checkpoint.

This novel apparent loss of Ade⁺ recombinants after UV irradiation observed with the checkpoint mutants could have occurred if for some reason Ade⁺ cells were more hypersensitive to UV irradiation than parental cells with the ade6^- heteroallelic duplication. For rad1, rad3, rhp9, chk1, and chk1 cds1 cells, we compared the UV sensitivity of parental ade6^- cells and Ade⁺ His^- deletion-type and Ade⁺ His⁺ conversion-type recombinants derived from the parental strains. There were no significant differences (P > 0.05) between UV sensitivities of the Ade⁺ cells and the corresponding parental cells for any of the checkpoint mutants tested (data not shown). Also, we seeded a suspension of chk1 ade6^- duplication parental cells with chk1 Ade⁺ His^- recombinant cells (10⁶–10⁷ ade6^- duplication cells/ml, 2 x 10³ Ade⁺ His^- cells/ml) and used it for a UV-induced recombinant assay. The preseeded Ade⁺ His^- cells still formed viable colonies on selective plates after UV irradiation, with the same UV sensitivity as Ade⁺ cells on the nonselective plates (data not shown). These results show that the fall in recombinant frequencies after UV observed for the checkpoint mutants is not due to greater UV sensitivity of Ade⁺ cells compared to parental ade6^- cells.

For cds1 cells, which lack only the intra-S damage checkpoint, compared to the frequency of spontaneous recombinants, there was no increase or fall in total Ade⁺ recombinant frequencies following UV irradiation (Fig 2A and Fig B). There was also no change in the relative proportion of deletion and conversion types (Fig 2B).

For rqh1 cells, which lack S-phase arrest survival/recovery, UV irradiation induced Ade⁺ recombinants at a higher frequency compared to wild-type cells and most of these induced recombinants were deletion types (Fig 2A and Fig B), as shown previously (DOE et al. 2000 ). Rqh1 appears to be necessary to suppress UV-induced recombination, particularly pathways that generate deletion-type recombinants.

UV irradiation does not result in loss of recombinants in a cds1 mutant even if cells are irradiated in G₁:
Since most cells used in these assays were likely to be initially in G₂, the effect of the loss of the intra-S checkpoint in cds1 cells may have been obscured. Recombination assays were therefore carried out with wild-type and cds1 cells initially synchronized in G₁ by nitrogen starvation. Wild-type cells damaged in G₁ exhibit an intra-S-phase delay while cds1 cells do not, as shown previously (LINDSAY et al. 1998 ; RHIND and RUSSELL 1998B ). For both wild-type and cds1 cells, spontaneous recombinant frequencies were unaffected by initial synchronization of cells in G₁ (Fig 3). The UV-induced recombinant frequencies of both wild-type and cds1 G₁ cells (Fig 3) were very similar to unsynchronized (mostly G₂) cells. For wild-type cells UV irradiation induced recombinants with a preferential induction of conversion-type recombinants. For cds1 cells, there was again no increase or decrease in the frequency of recombinants after UV irradiation compared to spontaneous frequencies and no change in the relative proportion of deletion- and conversion-type recombinants.

View larger version (24K): In this window In a new window Download PPT slide   Figure 3. Effect of UV irradiation on the frequency and distribution of recombinants in (A) wild-type and (B) cds1 cells initially synchronized in G1 of the cell cycle by nitrogen starvation. The cells were irradiated in G1 at UV doses of 0, 20, 40, 60, and 80 J/m2.

An artificial G₂ arrest allows recombinants to arise following UV damage in a G₂/M checkpoint mutant:
The cdc25-22 mutation was utilized to synchronize wild-type, chk1, and rad3 cells in G₂. We then determined whether a cdc25-imposed artificial 2-hr G₂ delay following UV irradiation affected recombinant frequencies compared with cells released from the cdc25 arrest immediately after UV irradiation. The results of these recombination assays are shown in Table 3. Spontaneous recombinant frequencies for all three strains were unaffected by initial synchronization of cells in G₂ (compare data in Table 2 and Table 3, 0 J/m² UV). In the absence of UV irradiation, recombinant frequencies were also unaffected by the additional cdc25-imposed 2-hr G₂ delay (Table 3, –G₂ and +G₂ data for 0 J/m² UV).

For cdc25-22 wild-type cells, UV irradiation induced recombinants as before, with a preferential stimulation of conversion-type recombinants, irrespective of whether cells were released from the cdc25 arrest immediately after irradiation or after 2 hr (Table 3). UV survival was also unaffected.

For cdc25-22 chk1 and cdc25-22 rad3 cells initially synchronized in G₂ but released from the cdc25-22 arrest immediately after UV irradiation, there was a fall in recombinant frequencies compared to spontaneous levels (Table 3, -G₂ data), as observed with unsynchronized cdc25⁺ chk1 and cdc25⁺ rad3 cells (Fig 2). Compared to cdc25-22 chk1 and cdc25-22 rad3 mutant cells allowed to reenter the cell cycle immediately after UV irradiation, those artificially delayed in G₂ for 2 hr were more UV resistant (Table 3, -G₂ and +G₂ survival data), as shown previously (AL-KHODAIRY and CARR 1992 ; AL-KHODAIRY et al. 1994 ). There were two possible outcomes for the recombination phenotype associated with this increased UV resistance. If the formation and/or viability of Ade⁺ recombinants in UV-irradiated chk1 and rad3 cells depends on a G₂ delay, then the recombinant frequency should also be elevated as well as UV survival. If, on the other hand, the formation and/or viability of Ade⁺ recombinants depend on a function of Chk1 and Rad3 other than their G₂/M checkpoint function, then the UV-induced recombinant frequency would not be elevated by an artificial G₂ delay. For both cdc25-22 chk1 and cdc25-22 rad3 cells, the increased UV survival of cells held in G₂ for 2 hr after irradiation was accompanied by an increase in recombinant formation (Table 3, -G₂ and +G₂ recombinant frequency data), although the level of recombinants still remained slightly below the level of spontaneous recombinants. These data indicate that a cell cycle delay allows enhanced levels of recombinant formation, but on its own is not sufficient to restore recombination to normal UV-induced levels.

UV sensitivity of cells synchronized in G₁ compared to unsynchronized (mostly G₂) cells:
S. pombe cells are more radiation resistant during the G₂ phase of the cell cycle than during G₁ (FABRE 1973 ). It was suggested that this was due to additional repair by homologous recombination between sister duplexes during G₂. We compared the UV resistance of cells synchronized in G₁ with unsynchronized, exponentially growing cells (70–80% in G₂) for wild-type, cds1, rad3, chk1, and chk1 cds1 strains. For wild type and cds1, G₂ cells were more UV resistant than were G₁ cells (Fig 4A and Fig B). In contrast, rad3, chk1, and chk1 cds1 G₂ cells were less UV resistant than were G₁ cells (Fig 4, C–E). Thus, it appears that the greater UV resistance of G₂ cells may depend on the G₂/M DNA damage checkpoint.

View larger version (19K): In this window In a new window Download PPT slide   Figure 4. UV sensitivity of cells synchronized in G1 compared to unsynchronized (mostly G2 cells. (A) Wild-type and (B) cds1 cells were more UV sensitive if irradiated in the G1 phase of the cell cycle compared to irradiation of mostly G2 cells. In contrast, (C) rad3, (D) chk1, and (E) chk1 cds1 cells were more UV sensitive if irradiated in the G2 phase of the cell cycle than if irradiated in G1.

Loss of recombinants is suppressed in rad3, chk1, and chk1 cds1 cells synchronized in G₁ prior to UV irradiation:
Since cells lacking the G₂/M checkpoint were more UV resistant in G₁, we decided to test whether the loss of viable recombinants following UV damage in G₂/M checkpoint mutants also occurred if cells were initially synchronized in G₁ prior to plating and irradiation. Synchronization of rad3, chk1, and chk1 cds1 cells in G₁ by nitrogen starvation had no effect on spontaneous recombinant frequencies compared to unsynchronized cells. However, although there was still a loss of recombinants following UV irradiation in all three mutants, the loss was more moderate than with irradiated unsynchronized cells (Fig 5). Thus the greater UV resistance of G₂/M damage checkpoint mutant cells irradiated in G₁ correlated with an increase in the frequency of viable recombinants following UV irradiation.

View larger version (13K): In this window In a new window Download PPT slide   Figure 5. Cell cycle phase dependence for level of loss of viable Ade+ recombinants following UV irradiation in (A) rad3, (B) chk1, and (C) chk1 cds1 cells. Compared to the dramatic loss of viable recombinants following UV damage of G2/M checkpoint mutant cells that were unsynchronized (mostly G2) prior to irradiation, UV irradiation of mutant cells synchronized in G1 results in enhanced viability of recombinants. Solid lines represent the effect of UV irradiation on recombinants for cells irradiated in G1. Broken lines represent the effect on recombinants of UV irradiating unsynchronized (mostly G2) cells, as obtained previously (Fig 2A). The G1 cells were irradiated at the following UV doses (J/m2): 0, 5, 10, 15, and 20 for rad3 and chk1 cds1 cells; 0, 15, 30, 45, and 60 for chk1 cells.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

There is mounting evidence for an intimate relationship between homologous recombination and cell cycle checkpoints in both yeasts and higher eukaryotes. We have sought to examine this relationship using the fission yeast model system. The most important finding of this study is that for G₂/M checkpoint mutants, both UV-induced recombinants and cells committed to spontaneous recombination are lost after UV-induced DNA damage.

Effect of checkpoint mutants on spontaneous mitotic intrachromosomal recombination:
Spontaneous mitotic recombination could reflect the recombinational repair of endogenous DNA damage (SWANSON et al. 1999 ). However, the role of the recombinational repair machinery as a housekeeping function in normal DNA replication is also becoming increasingly apparent (reviewed in HABER 1999 ; ROTHSTEIN et al. 2000 ; BENARD et al. 2001 ). It is envisaged that spontaneous mitotic recombination reflects the conversion of stalled replication forks into recombinogenic intermediates that allow replication restart (reviewed in HABER 1999 ; ROTHSTEIN et al. 2000 ).

The checkpoint mutations exhibited no effect (rad1, rad3, rad9, rad17, rhp9, chk1) or else a moderate effect (rad26, hus1; cds1, chk1 cds1) on spontaneous recombinant frequencies compared to wild-type cells (Table 2). In a previous study rad1-1 and rad3-136 mutations were also shown to have no effect on spontaneous recombination (FORTUNATO et al. 1996 ). The lack of major effects was not entirely surprising since S. pombe checkpoint mutant cells exhibit no perturbations in their cell cycle progression under normal growth conditions even though they still experience spontaneous DNA damage resulting from normal cellular metabolism. This infers that such spontaneous DNA damage and its associated repair/tolerance are silent to the checkpoint machinery.

The moderate (less than twofold) effect on spontaneous recombination exhibited by rad26 and hus1 cells, although statistically significant, may have been due to the "noise" associated with fluctuation tests for recombinant frequencies. A wealth of genetic and biochemical data place rad3⁺ and rad26⁺ in the same epistasis group, and the same goes for hus1⁺, rad1⁺, and rad9⁺. Epistasis studies with respect to recombination phenotype would be required to definitively determine whether the recombination effects seen in hus1 and rad26 cells are real.

The basis of the more marked (more than twofold) spontaneous hyper-recombination phenotype of cells lacking Cds1 is unknown. Rqh1 mutants also displayed a spontaneous hyper-recombination phenotype (Table 2; DOE et al. 2000 ). The hyper-recombination phenotypes may be related to their S-phase arrest survival/recovery function (MURRAY et al. 1997 ; STEWART et al. 1997 ; LINDSAY et al. 1998 ).

UV induces intrachromosomal recombinants in wild-type cells irradiated in G₁ and G₂:
For checkpoint-proficient wild-type cells, UV irradiation differentially induced deletion- and conversion-type recombinants. This suggests that recombination mechanisms, particularly those giving rise to conversion types, contribute to DNA repair/tolerance of UV damage and thus survival in wild-type cells. UV irradiation induced recombinants irrespective of whether irradiated cells were unsynchronized or prearrested in the G₁ or G₂ phases of the cell cycle. However, we cannot definitively say that UV-induced recombination events were initiated in G₁ and G₂. In S. cerevisiae, unexcised UV lesions induced in DNA of G₁ or G₂ cells have been shown to stimulate recombination during DNA replication (KADYK and HARTWELL 1993 ). Thus, UV lesions induced in G₁ and G₂ cells could remain unexcised and cause stalling of replication forks during S phase, resulting in elevated frequencies of recombinants.

In S. cerevisiae, UV damage incurred in G₂ cells also stimulated sister chromatid recombination directly in G₂ without the need for S phase (KADYK and HARTWELL 1993 ). Ade⁺ recombinants induced by irradiating G₂-synchronized wild-type cells (Table 3) may therefore also have been generated directly in G₂ without the need for DNA replication.

Loss of the intra-S-phase checkpoint results in loss of UV-induced recombinants, but not in the loss of viable spontaneous recombinants:
Loss of Cds1 function did not result in the loss of viable spontaneous recombinants following UV irradiation, unlike cells lacking the G₂/M DNA damage checkpoint. This was the case even when cds1 cells were irradiated in the G₁ phase of the cell cycle to ensure that the effect of the loss of intra-S-phase delay was not obscured.

However, cds1 cells lacked UV-induced recombinants. S. cerevisiae rad53 (presumptive homolog of cds1) mutant cells also lack UV-induced recombination (HAYNES and KUNZ 1981 ). The lack of UV-induced recombination could underlie the moderate UV sensitivity of cds1 cells at higher UV doses. It suggests that an intra-S-phase delay following UV damage is necessary to allow induced recombination. S. pombe cells lacking key recombination (rad22⁺, rhp51⁺, rhp54⁺) or nucleotide excision repair (NER; rad16⁺, swi10⁺) genes have a similar phenotype to cds1 cells: lack of UV-induced recombination (OSMAN et al. 2000 ). Thus, recombinational repair of UV lesions, either directly or after initial processing by excision repair pathways, may rely on the intra-S-phase delay mediated by Cds1. Interestingly, like cds1 cells, rhp51, rhp54, rad16, and swi10 cells also exhibit elevated spontaneous intrachromosomal recombination.

In S. cerevisiae Rad53 acts to stabilize DNA damage-stalled DNA replication forks (LOPES et al. 2001 ; TERCERO and DIFFLEY 2001 ). If UV-induced recombination reflects the recombinational rescue of replication forks stalled at UV lesions, a similar role for Cds1 would explain the lack of UV-induced recombination in cds1 cells. Alternatively, it could be due to additional defects of cds1 cells independent of their intra-S-phase checkpoint defect. It was shown that the FHA1 domain of Cds1 protein interacts with Mus81 protein, which appears to function in an Rhp51-dependent recombinational repair/tolerance pathway for UV damage (BODDY et al. 2000 ). Thus the lack of induced recombination in cds1 cells could be due to loss of a more direct role of Cds1 in homologous recombination.

The rqh1 mutant, which like the cds1 mutant lacks an S-phase arrest survival/recovery pathway in response to DNA damage (MURRAY et al. 1997 ; STEWART et al. 1997 ), did exhibit UV-induced recombination (Fig 2; DOE et al. 2000 ). The contrast in phenotypes between cds1 and rqh1 mutants suggests that the lack of UV-induced recombination in cds1 cells may not be due to an S-phase arrest survival/recovery defect, or that Cds1 and Rqh1 have distinct roles in this process.

UV irradiation causes the loss of viable mitotic recombinants in cells lacking the G₂/M DNA damage checkpoint:
The most important finding of this study is the observation that following UV irradiation, Ade⁺ recombinant frequencies for mutants lacking the G₂/M DNA damage checkpoint fell below spontaneous frequencies. To understand the significance of this novel phenotype, it is important to emphasize that Ade⁺ recombinants can arise after plating onto media selective for Ade⁺ recombinants, as well as during the initial growth of cells used in the assays. The generation of recombinants after plating is shown by the increase in recombinant frequency and the change in the relative distribution of deletion types to conversion types after UV irradiation of plated wild-type cells (Fig 2). The limited amount of intracellular adenine present in the plated cells could allow them to survive for a time and may be sufficient to allow a round of cell division. Indeed, stereo-zoom microscopic examination revealed that about 50% of wild-type and checkpoint mutant cells plated on media selective for Ade⁺ recombinants visibly divided once within 12 hr. The dramatic loss of viable recombinants in G₂/M damage checkpoint mutants following UV irradiation means that most recombinants must arise after plating on selective media.

The drop in recombinant frequencies after UV irradiation, to below spontaneous levels, suggests that cells undergoing recombination in the presence of UV damage, but without a G₂ delay, lose viability more frequently than do other cells. This is best understood by referring to Fig 6. For wild-type cells, Ade⁺ recombinants on the irradiated plates include both spontaneous recombinants and extra UV damage-induced recombinants. For cds1 cells lacking only the intra-S-phase delay, UV-induced recombinants are lost, but not spontaneous recombinants. For cells lacking the G₂/M checkpoint proteins, both spontaneous and UV-induced recombinants are lost.

View larger version (32K): In this window In a new window Download PPT slide   Figure 6. Significance of the recombination phenotypes of UV-irradiated cells. Most spontaneous and UV-irradiated Ade+ recombinants arise after plating. The recombinants on irradiated plates include both spontaneous recombinants, which arise due to endogenous DNA damage or intrinsic stalling of replication forks and extra UV-induced recombinants. (A) For UV-irradiated wild-type cells, Ade+ recombinants on the irradiated plates include both spontaneous recombinants (predominantly deletion types) and extra UV damage-induced recombinants (predominantly conversion types). The frequency of recombinants therefore increases after UV irradiation compared to spontaneous frequencies. (B) For cells lacking only the intra-S-phase delay (cds1 cells), UV-induced recombinants are lost but not spontaneous recombinants. Compared to spontaneous frequencies, therefore, there is no change in the frequency or distribution of deletion- and conversion-type recombinants following UV irradiation. (C) For cells lacking the G2/M checkpoint proteins, both spontaneous and UV-induced deletion- and conversion-type recombinants are lost following UV irradiation. The frequency of recombinants following UV irradiation therefore falls below the frequency of spontaneous recombinants. UV irradiation in the absence of G2/M checkpoint proteins therefore converts potentially viable spontaneous and UV-induced recombination events into lethal recombination events. *UV-induced recombinants graphs are frequency of recombinants plotted against fall in survival following UV irradiation.

Loss of viable mitotic recombinants after UV irradiation is associated with loss of the G₂/M checkpoint and is independent of the intra-S-phase delay:
Loss of recombinants after UV irradiation was observed in cells lacking the G₂/M checkpoint, whether or not they lacked only the G₂/M checkpoint (chk1 and rhp9), or both the G₂/M and intra-S-phase checkpoint (chk1 cds1 and rad checkpoint mutants). Therefore, loss of viability of recombinants following UV irradiation is independent of whether the intra-S checkpoint is intact or not.

The G₂/M checkpoint appears to be more critical to cell survival after UV damage than the intra-S-phase checkpoint since chk1 and rhp9 cells lacking just G₂ delay are more hypersensitive than cds1 cells lacking just the intra-S-phase delay, which are only moderately sensitive at high UV doses. This correlates with the loss of viable recombinants after UV in chk1 and rhp9 cells, whereas absence of Cds1 results only in abrogation of the extra UV-induced recombinants rather than the enhanced loss of viability of cells in the process of executing spontaneous recombination (Fig 6).

UV irradiation caused loss of recombinants in G₂-synchronized rad3 and chk1 cells allowed to reenter the cell cycle immediately after UV (Table 3). This suggests that loss of viability of recombinants may not require DNA replication during S phase and may thus be unrelated to recombinational rescue of stalled replication forks. However, this cannot be stated definitively since cells on selective plates may undergo cell division and DNA replication before losing viability, although this is unlikely for G₂/M checkpoint mutants UV irradiated in G₂.

What is the underlying cause for the loss of viable recombinants in UV-irradiated G₂/M checkpoint mutants?
The loss of viability associated with recombination can be explained in several ways, which are not mutually exclusive. One explanation is that a subset of plated cells are recombination proficient and, in these checkpoint mutants, they are for some reason much more UV sensitive or repair deficient than other nonrecombination-proficient cells.

Alternatively, after UV damage, successful homologous recombination, whether spontaneous or UV-induced, becomes dependent on a function of the G₂/M checkpoint proteins. That is, UV damage in the absence of a G₂/M protein function can transform a potentially viable recombination event into an aberrant one that results in cell inviability.

What might cause a potentially viable recombination event to become a lethal one in UV-irradiated G₂/M checkpoint mutants? Several mechanisms can be postulated:

1. Lack of the G₂ delay after DNA damage might result in lack of time for the completion of recombination events, leading to lethal recombination intermediates and cell inviability. This would be true for damage-induced recombination events. However, cells committed to spontaneous intrachromosomal recombination are also lost even though the time in G₂ available to generate a spontaneous recombinant after UV is not any shorter than in nonirradiated, nondelayed cells.

2. Lack of the G₂ delay might provide insufficient time for the activation/expression of recombinational repair genes. Again, this may not be sufficient to explain loss of spontaneous recombinants.

3. The biased loss in viability of cells that would have, in the absence of UV, formed spontaneous recombinants could be due to the titration of critical recombination proteins from these sites due to an excess of UV-damaged sites.

4. Loss of G₂/M checkpoint function, either damage-induced cell cycle delay or possibly a step in catalyzing some stage of recombination, may result in channeling of spontaneous and UV-induced DNA lesions into abortive recombination pathways or into other irreparable DNA structures.

5. The role of the G₂/M delay may be to allow time to ensure pairing and recombination between a damaged template and an appropriate homologous partner, probably the sister chromatid (KADYK and HARTWELL 1992 ). In response to DNA damage, S. cerevisiae rad9 checkpoint-defective mutants exhibit increased frequencies of translocations by inappropriate recombination between ectopically located homologous sequences and reduced frequencies of appropriate sister chromatid exchange (FASULLO et al. 1998 ). Correct partner choice during meiotic recombination in S. cerevisiae also requires proteins central to mitotic DNA damage checkpoint functions (GRUSHCOW et al. 1999 ; THOMPSON and STAHL 1999 ).

At present we cannot distinguish among these various possibilities for the mechanism for loss of viable recombinants. Herein we will refer to the mechanism that leads to inviability of recombinants as aberrant recombination. There is accumulating evidence that aberrant recombination can lead to loss of cell viability, particularly in DNA damaged cells lacking checkpoint delays. For example, it appears that aberrant homologous recombination is responsible for the severe effects of irradiation on chicken cells in S-G₂ when both checkpoint functions and nonhomologous recombination are defective (MORRISON et al. 2000 ). In a study that did not rely on cell survival to score for homologous intrachromosomal recombination, irradiated G₂/M cultured monkey cells that attempted recombination were much more inviable than those that did not (CAO et al. 1997 ). It appears that lethal levels of aberrant recombination cause the dramatic reduction in cell viability of S. cerevisiae srs2 sgs1 double mutants, since they can be suppressed by mutation of RAD51, RAD52, RAD55, and RAD57 (GANGLOFF et al. 2000 ; KLEIN 2001 ), which all act in the initial stages of homologous recombination to promote strand exchange.

Evidence in S. pombe (DOE et al. 2000 ) suggests that an excess of potentially lethal recombination intermediates in cells that lack the anti-recombinase activity of Rqh1 may be the cause of the defects observed in rqh1^- cells. These include loss of viability associated with hyper-recombination and problems with chromosome segregation, both spontaneous and after UV irradiation. The synthetic lethality of cells simultaneously lacking rqh1⁺ and some checkpoint functions (MURRAY et al. 1997 ) may be due to lethal aberrant recombination even in the absence of extrinsic DNA damage. The Mus81 protein may also be important for removing Holliday junctions that arise spontaneously and after UV irradiation in S. pombe (BODDY et al. 2000 ). Interestingly mus81 rqh1 double mutants are inviable and mus81 mutations trigger a Rad3-dependent checkpoint delay in the absence of extrinsic DNA damage (BODDY et al. 2000 ). The accumulation of unresolved Holliday junctions could be the underlying factor for the inviability of G₂/M cells undergoing recombination after UV irradiation. However, we tested the effect of a nuclear-targeted RusA Holliday junction resolvase (DOE et al. 2000 ) in chk1 cells and found that expression of RusA did not complement the hypersensitivity to UV, nor did it suppress the loss of viability of recombinants after UV irradiation (data not shown). DNA double-strand breaks could be an alternative potentially lethal recombination intermediate, but neither rad1-1 nor rad3-136 mutant S. pombe cells were defective in intrachromosomal recombination induced by a site-specific double-strand break within the intrachromosomal recombination substrate, and such a break did not result in a cell cycle delay in wild-type cells (FORTUNATO et al. 1996 ).

An artificial G₂ delay partially rescues the viability of recombinants after UV damage:
An essential role of the G₂/M transient arrest in S. pombe may be to allow enough time before mitosis specifically for the completion of homologous recombination events, both spontaneous and UV induced, to enable viable segregation of chromosomes. However, although a major function of the checkpoint proteins is to mediate cell cycle delays in response to damage, we now know from studies in S. pombe, S. cerevisiae, and mammalian cells that the checkpoint proteins do much more than just enforce cell cycle delays (reviewed in WEINERT 1998 ). In mammalian cells ATM appears to have an important role in controlling homologous recombination (reviewed in VAN GENT et al. 2001 ), but it still remains to be resolved whether this is due to the effect of ATM on cell cycle checkpoints or more directly on DNA repair. In S. cerevisiae DNA checkpoint proteins have also been shown to mediate DNA damage-induced transcription of many genes, including recombinational repair genes (ZHOU and ELLEDGE 1993 ; ALLEN et al. 1994 ; ELLEDGE 1996 ), and some appear to be directly involved in DNA metabolism or repair (LYDALL and WEINERT 1995 ). Checkpoint-dependent transcriptional activation in response to DNA damage has been observed in S. pombe, including the recent finding that UV damage-induced transcription of the recombination gene rhp51⁺ was significantly reduced in some checkpoint mutants (SHIM et al. 2000 ).

It was therefore important to determine whether the loss of recombinants following UV irradiation in the G₂/M checkpoint mutants was due to their inability to transiently arrest cells or whether it was due to their additional repair defects, such as the reduced ability to induce the key recombination gene rhp51⁺. This was achieved by imposing a 2-hr G₂ arrest on UV-irradiated rad3 and chk1 cells. As shown (Table 3), the loss of recombinants following UV irradiation could be partially compensated by imposing an artificial G₂ arrest on rad3 and chk1 cells. Loss of recombinants is therefore due, at least in part, to the absence of the G₂/M delay in cell cycle progression in response to UV damage. The lack of complete compensation suggests that loss of recombinants is also due partly to the additional defects of the G₂/M checkpoint mutants, independent of their defect in G₂ delay. Alternatively, this could have been due to the inadequacy of the experimentally imposed G₂ delay compared to the intact checkpoint-mediated delay.

UV damage in G₁ cells results in a decreased loss of viable recombinants:
Wild-type and cds1 cells initially synchronized in G₁ were less UV resistant than were cells predominantly in G₂ (Fig 4), and initial cell cycle phase had no effect on the frequency of Ade⁺ recombinants after UV irradiation. In contrast, rad3, chk1, and chk1 cds1 cells initially synchronized in G₁ were more UV resistant than were cells predominantly in G₂ (Fig 4; FABRE 1973 ; AL-KHODAIRY et al. 1994 ). The increased resistance of G₁-irradiated cells correlated with improved recovery of viable recombinants after UV irradiation in these G₂/M checkpoint mutants (Fig 5). This could account for the cell cycle-specific differences in sensitivity of these G₂/M checkpoint mutants. DNA damage-dependent phosphorylation of Chk1 has also been shown to be cell cycle specific, occurring primarily in late S phase and G₂, but not during M/G₁ or early S phase (MARTINHO et al. 1998 ). In S. cerevisiae most G₁-induced UV lesions appear to be removed by NER prior to the onset of DNA replication (KADYK and HARTWELL 1993 ). UV-induced lesions could also be excised in S. pombe cells synchronized and maintained in G₁ (OSMAN and MCCREADY 1998 ). Thus, the decreased loss of recombinants after UV damage and the greater UV resistance of G₂/M checkpoint mutants irradiated in G₁ could be due to repair of UV lesions via nonrecombinational pathways. In contrast, recombinational repair may predominate in cells damaged in S phase or G₂, which in the absence of a G₂/M checkpoint delay results in aberrant recombination causing cell inviability.

Summary:
In this study we have discovered a novel recombination phenotype for G₂/M checkpoint mutants. The phenotype can best be explained as follows: UV damage in the absence of a G₂ delay causes both spontaneous and UV-induced recombination events to become aberrant, leading to cell inviability. This loss of viable recombinants in G₂/M checkpoint mutants (1) is independent of the intra-S-phase checkpoint, (2) may not require DNA replication, (3) cannot be suppressed by expression of a prokaryotic Holliday junction resolvase, (4) is partially suppressed by an experimentally imposed G₂/M checkpoint, and (5) is suppressed by irradiating cells in G₁, which correlates with the increased resistance to UV of G₁ cells compared to G₂ cells. As well as the lack of extra time for repair, aberrant recombination in the absence of a G₂ delay may be an underlying cause of the hypersensitivity to DNA-damaging agents of G₂/M checkpoint mutants.

The aberrant recombination responsible for loss of viability of recombinants is likely to rely on the catalytic activity of DNA repair or recombination proteins. To determine which proteins may be involved we are testing to see if the loss of recombinants can be suppressed by additional mutation of genes involved in these processes. It would also be interesting to determine whether overexpression of Rqh1, an anti-recombinase, might also suppress the loss.

	ACKNOWLEDGMENTS

We thank Tony Carr (Sussex University, UK) for providing the original checkpoint mutant strains. Thanks are also due to Tony Carr, Alan Lehmann, and their colleagues (Sussex University) for useful discussions and ideas during the progress of this work. This work was funded primarily by a Wellcome Trust project grant (054358/BS/JS) awarded to I.T. and F.O. and also by Wellcome Trust Senior Research Fellowships awarded to I.T. and M.W.

Manuscript received October 2, 2001; Accepted for publication December 14, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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