Checkpoint Arrest Signaling in Response to UV Damage Is Independent of Nucleotide Excision Repair in Saccharomyces cerevisiae*

Hong Zhang, Jena TaylorDagger, and Wolfram Siede§

From the Department of Radiation Oncology and the Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322

Received for publication, January 3, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The recognition of DNA double-stranded breaks or single-stranded DNA gaps as a precondition for cell cycle checkpoint arrest has been well established. However, how bulky base damage such as UV-induced pyrimidine dimers elicits a checkpoint response has remained elusive. Nucleotide excision repair represents the main pathway for UV dimer removal that results in strand interruptions. However, we demonstrate here that Rad53p hyperphosphorylation, an early event of checkpoint signaling in Saccharomyces cerevisiae, is independent of nucleotide excision repair (NER), even if replication as a source of secondary DNA damage is excluded. Thus, our data hint at primary base damage or at UV damage (primary or secondary) that does not need to be processed by NER as the relevant substrate of damage-sensing checkpoint proteins.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Checkpoints arrest cell cycle progression in the presence of DNA damage to facilitate DNA repair and prevent genetic instability (1-3). There is now a considerable amount of information available that characterizes signal transmission mechanisms and downstream targets in lower and higher eukaryotic cells. However, the precise roles of the initial damage-sensing proteins and their relevant DNA or chromatin substrates have remained largely elusive. Characterization of candidate proteins supports the notion that the underlying mechanisms are conserved throughout eukaryotic evolution (4-7). Consequently, the yeast Saccharomyces cerevisiae will continue to play an important role as a relevant model system.

A damage sensor function has been suggested for the PCNA1-like complex composed of the budding yeast proteins Rad17p, Ddc1p, and Mec3p, also termed 9-1-1 complex (following the nomenclature adapted for human cells and Schizosaccharomyces pombe) (8-11). Although direct evidence is still missing, it is likely that this ring-shaped complex (12) encircles DNA and is loaded onto DNA by a replication factor C-related complex in which the largest replication factor C subunit is replaced by the checkpoint protein Rad24p (13). The phosphoinositol kinase-related protein Mec1p (an ortholog of the human ATR protein) has been identified as an independent damage-sensor candidate (9, 11). Mec1p forms a complex with Lcd1p, a protein with an affinity for DNA double-stranded breaks, and thus Mec1p kinase activity can be targeted to sites of DNA damage (14). Mec1p and its downstream target Rad53p are involved in virtually all DNA damage and replication- related checkpoints.

Other potential sensor proteins responding to double-stranded breaks are represented by the Rad50p·Xrs2p·Mre11p complex. This complex plays a well established role in non-homologous endjoining and homologous recombination, whereas a function in checkpoint arrest following treatment with strand break-inducing agents has only recently been uncovered (15, 16).

Although such putative damage recognition complexes appear to be formed independently from each other, their combined action is frequently necessary for the generation of a downstream signal. A key player in signal transmission is the Rad53p kinase that is phosphorylated in a Mec1p-dependent fashion in response to DNA damage (17). A pool of hyperphosphorylated Rad53p is generated through autophosphorylation in trans, mediated through complex formation with Rad9p following Mec1p-dependent Rad9p phosphorylation (18).

In higher and lower eukaryotes, several studies have indicated that even a single DNA double strand break can result in checkpoint activation if not immediately repaired (19-21). Other observations point to the significance of single-stranded DNA tracts (21, 22). Surprisingly however, little is known regarding the recognition of base damage that is not directly associated with double strand breaks, even for such a generally well studied case as UV damage. Exposure of DNA to ultraviolet light (mainly UV-C) results in the dimerization of adjacent pyrimidine residues, giving rise to cyclobutane-type dimers and 6-4 photoproducts (23). These types of bulky DNA damage are readily recognized and repaired by nucleotide excision repair (NER), which functions by the introduction of single strand breaks bracketing the lesion, followed by the removal of a single-stranded DNA fragment (24). A global NER system and a repair system that is targeted by inhibition of transcription to the site of the lesion can be distinguished. The established correlation of double-stranded breaks and possibly single-stranded DNA with checkpoint arrest could hint at a NER intermediate involving free DNA ends as the effective recognized lesion following UV treatment. However, any single-stranded gaps arising directly from NER will be small and short-lived.

In S. cerevisiae, two studies have analyzed in some detail checkpoint responses at the G1/S transition in NER-deficient cells (25, 26). An S-phase delay was observed in UV-irradiated NER-deficient cells at much lower doses than in the isogenic wild type (25). However, this delay was not influenced by Rad9p, a protein necessary for both G1 and G2/M checkpoint arrest in the wild type. More detailed analysis indicated that UV-irradiated NER-deficient cells do not arrest upstream of START as the wild type, albeit at higher doses (25). At the molecular level, it was independently confirmed that this low dose arrest response observed in NER-deficient cells depends on S-phase entry and requires the checkpoint kinases Mec1p and Rad53p but not other checkpoint proteins including putative damage sensors (such as Rad9p, Rad17p, Rad24p, Mec3p, or Ddc1p) (26). Nevertheless, these proteins are needed for efficient UV-induced mutagenesis in NER-deficient cells, and a subset is also required for UV-induced sister chromatid exchange (27).

These observations neither prove nor disprove the view that damage processing by NER is indispensable for checkpoint responses to UV damage outside of S-phase. It needs to be taken into account that the effects analyzed so far have been triggered by much lower doses of UV than usually given to wild-type cells. If primary unprocessed UV damage is the relevant trigger of checkpoint signaling, the dose range required for wild-type responses will have to be applied. However, any interpretation will have to consider the high lethality of NER mutants in such dose range.

This study addresses primarily the requirement of NER for events that precede cell cycle checkpoint arrest. While avoiding complications because of secondary lesions arising from the interference of photodimers with replication, we have concentrated on the analysis of NER-deficient cells that were synchronized and UV-treated in G1- and M-phase. We conclude that in these cell cycle stages NER is not required for Rad53p hyperphosphorylation, an indispensable step of arrest signal transmission. However, such an independence of NER is only uncovered when approximately the same UV dose range is applied that is required for Rad53p activation in the wild type. Although the resulting high lethality ultimately precludes a meaningful interpretation of the actual cell cycle effects, our data support models that assume recognition of UV-induced initial base damage and not of NER-dependent DNA repair intermediates by sensor proteins as the essential step. Alternatively, primary or secondary non-dimer DNA damage may be critical as recognized lesions.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Strains-- The haploid S. cerevisiae strain SX46A MATa ade2 ura3-52 trp1-289 his3-532 was used as wild type (source: E. C. Friedberg, originally from J. Rine). In this strain background, a pronounced G1 arrest following treatment with UV or ionizing radiation is observed (28). Construction of the NER-deficient rad14Delta ::HIS3 and rad9Delta ::URA3 derivatives have been described elsewhere (25, 28). A cdc4 mutant (WS9110-3D MATa cdc4-1 ade ura3-52 trp1-289) was derived from the same background by crossing (25). In this strain, a RAD14 deletion was introduced through homology-mediated trans-placement with a rad14Delta ::KanMX4 PCR fragment that was originated from an existing null strain obtained from a yeast strain repository (Euroscarf).

Cell Synchronization and UV Treatment-- For cell culture and media recipes, we followed standard procedures of yeast genetics and cell biology (29). Cells were synchronized in M-phase by resuspending a mid-logarithmic phase culture in fresh YPD (1% yeast extract, 2% peptone, 2% dextrose) at 1 × 107 cells/ml and by adding nocodazole (U. S. Biological) to 10 µg/ml. A second aliquot was added after 60 min of incubation at 30 °C, and the culture was used following another 75 min of incubation. Cells were synchronized in G1 with the yeast pheromone alpha -factor (U. S. Biological). A mid-logarithmic phase culture (~1 × 107 cells/ml) was resuspended in fresh YPD, and alpha -factor was first added to 10 µg/ml. A second aliquot was added after 75 min of incubation at 30 °C or after 90 min at 25 °C in the case of cdc4 strains. Synchronization in G1 was complete after another 75 or 90 min in the case of cdc4 strains. Synchronized cells were washed free of medium, resuspended in deionized water at a titer of 2.5 × 107 cells/ml, sonicated briefly, and kept on ice. 15- or 30-ml samples of the cell suspension were irradiated under constant stirring in 100- or 150-mm-diameter plastic Petri dishes, respectively. The used lamp delivered 254 nm of UV-C at a dose rate of 1 or 2 J/m2 × s- (Sanyo Denki Germicidal Lamp G15T8). Appropriate dilutions were plated on solid YPD to determine survival. Pelleted, irradiated, and mock-treated cells were resuspended in fresh YPD at a titer of 1 × 107 cells/ml. Samples were taken during subsequent incubation at 30 °C with the exception of cdc4 mutant cells, which were kept in G1 by incubation at 37 °C. If required, alpha -factor-synchronized strains were kept arrested in G1 following irradiation by resuspension in their old pheromone-containing medium and by the addition of further aliquots of alpha -factor immediately and after 1 h of incubation.

Cell Cycle Analysis-- For FACS analysis, ~1 × 107 cells were withdrawn at each time point, spun down, washed, and fixed in 90% ethanol. Staining with propidium iodide has been described elsewhere (30). Samples were analyzed on a BD Biosciences FACScan using the CellQuest program suite.

Detection of Rad53p-- A sample of 1 × 108 cells was used for each time point. Trichloroacetic acid protein extracts were prepared using zirconium beads (Biospec Products) essentially as described (31). Approximately one-third of total protein extract was loaded per lane of an 8% PAGE gel (1:80 bisacrylamide:acrylamide ratio). Separated proteins were transferred to nitrocellulose by semidry electroblotting, and immunoblot analysis was performed according to standard techniques (32). S. cerevisiae Rad53p antibody (Sc6749, raised in goat, Santa Cruz Biotechnology) was used at a 1:300 dilution, secondary anti-goat horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology) at a 1:3000 dilution. SuperSignal West Pico kit (Pierce) was used for chemiluminescence signal detection.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Cycle Response in UV-Treated M-phase Cells-- In S. cerevisiae, the influence of NER on UV-induced cell cycle arrest and associated responses has not been investigated in cell cycle stages other than G1/S. We regarded studying UV-treated M-phase cells as an attractive approach because a checkpoint response can be determined that is not influenced by the generation of secondary DNA damage in S-phase. To this end, cells synchronized in metaphase by treatment with the tubulin inhibitor nocodazole were UV-irradiated in suspension and released in fresh medium without inhibitor. The macrocolony survival resulting from this and all further treatment protocols is shown in Fig. 1. Cell cycle progression was analyzed by FACS analysis. In repair-competent wild-type cells, a dose of 20 J/m2 resulted in only a brief M-phase arrest during the first cycle, whereas treatment with 120 J/m2 caused an arrest of at least 2 h (Fig. 2A). In NER-deficient rad14Delta cells, a similar brief arrest was found at the lower dose (Fig. 2B). In contrast to the wild type, this M-phase arrest was followed by a very pronounced arrest in a stage with an apparent G1 DNA content. For the higher dose, the NER-deficient cells remained arrested with a G2/M DNA content throughout the experiment (Fig. 2B) and never resumed cell cycle progression (data not shown). In the checkpoint and NER-deficient rad9Delta rad14Delta double mutant, essentially the same results were found apart from a somewhat prolonged M-phase arrest at the lower dose (Fig. 2C). However, it is important to note that these experiments were performed with lethally damaged cells (Fig. 1). For the lower UV dose, the colony survival of both single mutant (rad14Delta ) and double mutant (rad14Delta rad9) was already <0.01% (Fig. 1). Therefore, it is difficult to interpret the observed cell cycle effects as actively regulated, physiological checkpoint responses.


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Fig. 1.   Macrocolony survival of haploid yeast cells treated with UV-C in M-phase or in G1-phase. Cells were synchronized with nocodazole (filled symbols) or with alpha -factor (open symbols). The strains used were SX46A RAD (black-square, ); rad14Delta (, open circle ) or rad14Delta rad9Delta (black-diamond , diamond ); and WS9110-3D cdc4-1 RAD (down-triangle) or cdc4-1 rad14Delta (triangle ).


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Fig. 2.   Cell cycle distribution in cells synchronized in metaphase by nocodazole and released from arrest following UV-C treatment. Samples were withdrawn at the times indicated to the left following irradiation with 0, 20, or 120 J/m2 UV, and DNA content was detected by FACS analysis. The strains used were SX46A RAD, rad14Delta , and rad14Delta rad9Delta .

Rad53p Hyperphosphorylation in UV-Treated M-phase Cells-- We reasoned that a rapid signaling response such as Rad53p hyperphosphorylation that is more proximal to the damage recognition process might be less affected by the lethally damaged state of the studied mutant cells. Thus, we analyzed in the samples shown in Fig. 2 the extent of Rad53p hyperphosphorylation in the form of slower moving protein bands visualized by immunoblotting (Fig. 3). Following treatment with the lower UV dose (20 J/m2), only a small fraction of Rad53p was found to be transiently phosphorylated, whereas the higher dose resulted in a very pronounced Rad53p modification. Most importantly, these results were essentially the same in both the wild type and NER-deficient rad14 mutant (Fig. 3). The only difference between the strains appeared to be the diminishing level of hyperphosphorylated Rad53p that is observed after 3 h of incubation in the wild type but not in the mutant. Thus, the phosphorylation level correlates with exit from M-phase arrest (Fig. 2) and may reflect successful DNA damage repair. As expected, UV-induced Rad53p hyperphosphorylation was found to be strictly dependent on RAD9 (Fig. 3).


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Fig. 3.   Rad53p detection by immunoblotting in cells synchronized in metaphase and released from arrest following UV-C treatment. Samples were taken at the times indicated at the top of the lanes following irradiation with 0, 20, or 120 J/m2 UV. The cell cycle distribution of each sample was as shown in Fig. 2.

Rad53p Hyperphosphorylation in UV-treated G1-arrested Cells-- Because these data indicate that Rad53p phosphorylation is not significantly influenced by the status of NER in UV-irradiated M-phase cells, we decided to re-evaluate the influence of NER on checkpoint signaling in G1 cells over a range of UV doses. We synchronized the identical strains with the yeast pheromone alpha -factor in G1, treated with UV, and continued incubation in the presence of alpha -factor to exclude replication as a source of secondary DNA damage (Fig. 4). Microscopic examination indicated continued "shmoo" appearance of the cells, and FACS analysis confirmed G1 DNA content throughout the experiment (Fig. 4B). As with M-phase cells, we determined that the extent and dose response of UV-induced Rad53p hyperphosphorylation were quite similar in wild type and rad14Delta mutant cells (Fig. 4A). Especially at the lower dose, the mutant strain appeared to have a more pronounced response than the wild type. Again, Rad53p modification was dependent on functional Rad9p (Fig. 4A).


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Fig. 4.   Rad53p phosphorylation in cells synchronized and kept arrested in G1 with alpha -factor following UV-C treatment. A, Rad53p was detected by immunoblotting in cells irradiated with 0, 10, or 60 J/m2 UV as indicated. Samples were taken at the times noted at the top of the lanes. The strains used were SX46A RAD, rad14Delta , and rad14Delta rad9Delta . B, FACS profiles of 0- and 180-min samples indicate continued arrest in G1.

Additionally, we prevented S-phase entry in G1-synchronized UV-treated cells by the use of the thermoconditional cdc4-1 mutation (Fig. 5). RAD cdc4-1 and rad14Delta cdc4-1 cells were treated with alpha -factor at permissive temperature, irradiated with a range of UV doses, and incubated at 37 °C to block cell cycle progression and entry into S-phase. Continuous arrest in G1 was confirmed by FACS analysis (data not shown). With the low doses of 4 and 8 J/m2, we observed some degree of Rad53p hyperphosphorylation at the later time points in the NER-deficient strain but not in the NER-proficient wild type. With the higher doses, there was essentially no major difference in the extent and time course of the observed Rad53p hyperphosphorylation between the strains (Fig. 5). Indicating successful repair, a reappearance of the original non-modified Rad53p band was noted after 3 h of incubation in the NER-proficient strain but not in the NER-deficient strain (20 and 40 J/m2).


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Fig. 5.   Rad53p phosphorylation in cdc4 cells synchronized in G1 with alpha -factor and kept in G1 by incubation at the restrictive temperature following UV-C treatment. Cells were kept arrested at 37 °C following treatment with 4, 8, 20, 40, and 60 J/m2 as indicated. Samples were taken at the times indicated at the top of the figure. Strains WS9110-3D cdc4-1 RAD and cdc4-1 rad14Delta are compared.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A considerable amount of information is available on the highly sensitive and specific initiation of cell cycle checkpoint arrests and associated responses following the occurrence of DNA double-stranded breaks (1-7). Surprisingly, little is known regarding the initiating molecular events that accompany such responses in cells exposed to base-damaging agents that by themselves do not introduce DNA double-stranded breaks or single-stranded gaps. Here, we have concentrated on the analysis of the effects of UV-C because the nature of the induced base damage (i.e. pyrimidine dimers) and its repair are well understood (23, 24). Ultraviolet radiation is a potent trigger of checkpoint arrest and transcriptional responses in lower and higher eukaryotes (23, 33-36).

At the outset, several mechanisms of damage recognition can be envisioned. First, a specific type of primary DNA lesion may be directly recognized by the binding of one or more sensor proteins. Second, the conversion of primary damage into a DNA repair intermediate or the formation of a DNA-repair protein complex may create a substrate for damage-sensing components of the checkpoint pathways. Third, the interference of damage with cellular processes such as DNA replication or transcription may elicit the response. This may include the formation of secondary structural DNA damage such as single-stranded gaps during DNA synthesis. Lastly, the possibility should not be dismissed that non-DNA targets of the applied agent play essential roles in checkpoint activation. The identification of protein kinase p38 as an important component of UV-induced G2 arrest in mammalian cells may provide such an example (37).

Because no data on G2/M-phase regulation in NER-deficient budding yeast were available, we first characterized UV-induced M-phase arrest in cells synchronized at the metaphase/anaphase transition. In response to DNA double strand breaks, a bifurcated pathway regulates checkpoint arrest at or downstream of this stage with both branches being dependent on Mec1p kinase (38-41). One subpathway regulates the securin Pds1p through the Chk1p kinase, whereas the other involves the kinases Rad53p and Dun1p. However, Chk1p appears be dispensable for cell cycle arrest after UV (39, 42), and it remains to be demonstrated that the arrest pathways for UV damage are similarly organized.

In this study, we found that a significant dose-dependent M-phase arrest can be induced by UV irradiation in cells released from nocodazole arrest. Rad53p hyperphosphorylation correlates well with the arrest response in the wild type. A similar analysis in a rad14Delta mutant revealed that the extent, dose dependence, and time course of Rad53p modification remain essentially unaffected by a NER defect. The Rad14 protein is necessary for early steps of UV damage recognition, and deletion mutants are completely deficient in the incision step (43, 44). We further demonstrated that Rad9p is clearly required for UV-induced Rad53p phosphorylation, even in the NER- background.

Deliberately, we have not attempted to modify the UV dose according to the much lower survival level of NER-deficient cells. However, we have to consider the implications of working with a population of lethally damaged cells that are most probably severely inhibited in all cellular functions (such as transcription) shortly after treatment. A recent study (45) has suggested a defect in UV-induced G2/M arrest in NER-deficient cells solely based on budding analysis. However, we doubt that a morphological marker is meaningful in lethally irradiated cells. We argue that the observed early marker for checkpoint activation, Rad53p hyperphosphorylation, represents a physiological response to UV damage even in NER-deficient cells because the extent and time course do not appear to be different from wild type. We are less certain regarding the observed cell cycle effects. NER-deficient cells lethally irradiated with 20 J/m2 are still capable of M/G1 transition following a delay in M-phase. However, this delay is not affected by an additional rad9 defect and thus represents more likely a passive inhibitory effect of damage than an actively regulated checkpoint response. Alternatively, a different RAD9-independent checkpoint may have been triggered. A high level of unrepaired DNA damage in mitosis may activate the spindle-assembly checkpoint as described recently for human cells (46). In our experiments, NER-deficient M-phase cells lethally irradiated with 120 J/m2 remained and ultimately died in this stage.

Our observations suggest that checkpoint signaling through the Rad53p pathway is independent of UV dimer incision and, thus, that a damage-recognition mechanism that targets primary DNA base damage and not repair-induced strand interruptions may be operating. However, although the FACS profiles argue against massive DNA degradation, we have not formally demonstrated the absence of DNA strand breakage. Certain double-stranded break repair-deficient mutants in S. cerevisiae exhibit a pronounced UV sensitivity, and double-stranded breaks are clearly detectable during post-irradiation growth of UV-treated yeast (47). Throughout our experiments, S-phase is never entered and cannot be a source of secondary damage, but other mechanisms are not excluded. Double-stranded breaks may arise from recombinational repair of unrepaired UV damage in M-phase-synchronized cells. In yeast and other systems, UV-induced recombination events were indeed found to be independent of NER and replication (48-50). However, such an explanation can hardly account for our observations in haploid cells arrested in G1 where no homologous chromosome is available.

The apparent lack of effect of NER on checkpoint pathway activation prompted us to re-evaluate previous observations in G1 cells. An S-phase delay has been reported in NER-deficient strains at a much lower dose level than in the wild type, and this response turned out to be the result of RAD9-independent activation of a MEC1-dependent checkpoint response in early S-phase, most probably triggered by secondary damage or inhibition of replication fork progression (25, 26). However, G1-checkpoint arrest following treatment with DNA-damaging agents occurs at or upstream of START in repair-proficient cells (25, 45, 51). The influence of NER remained to be analyzed at a dose level required for such a response in the wild type. Obviously, the entry into S-phase had to be prevented. Thus, we confined the analysis of Rad53p phosphorylation to UV-treated cells that were kept arrested by alpha -factor or by incubation of cdc4-1 strains at the restrictive temperature. In both cases, UV-induced hyperphosphorylation of Rad53p was readily detectable. This is in contrast to studies indicating the inability of a single persistent double-stranded break to trigger Rad53 hyperphosphorylation in G1 cells (20). Most importantly, the independence of UV-induced Rad53p phosphorylation from NER was again observed.

Our data on cdc4-1 are in conflict with a previous study (26) that demonstrated in a comparable approach a need for S-phase entry for Rad53p hyperphosphorylation in NER-deficient strains but not in the wild type. Technical details of UV application cannot be compared directly between the two studies. However, we were able to verify our results over a range of doses. Differences in the used antibody and the strain background could provide possible explanations for the contrasting findings.

In summary, our results are compatible with models of UV damage recognition for checkpoint activation outside of S-phase that do not rely on the main pathway of dimer repair or on cell cycle progression. These observations are consistent with two types of hypotheses. First, the recognized DNA damage that triggers the checkpoint response is primary DNA damage such as UV-induced pyrimidine dimers that are recognized by checkpoint sensor proteins independent of NER. The extent of the response will depend on the level and persistence of initial damage and thus initially on the dose but subsequently also on cellular repair capacity. The continued presence of unrepaired damage may sensitize the response, and indeed, Rad53p phosphorylation is found in G1 cells at lower doses than in the wild type. However, this latter response is delayed and would not have prevented S-phase entry if cells had not been kept artificially in G1. Such a concept of initial damage recognition is supported by recent data on human ATR protein, the homolog of yeast Mec1p (52). The observed increased affinity of ATR protein for UV-dimer containing DNA indicates a role as a sensor of primary UV damage.

Alternative hypotheses will rely on UV-induced DNA damage that is not influenced by NER as the type of lesion relevant for checkpoint activation. Such damage could constitute non-dimer type primary damage or, alternatively, secondary lesions that originate from the interference of damage with cellular processes. These include transcription or non-NER repair, but our approach excludes an influence of replication. Such damage may in fact be identical to persistent double-stranded breaks, already known to be recognized by putative damage sensor proteins of the checkpoint pathways. However, one established double-stranded break sensor, the Rad50p·Xrs2p·Mre11p complex, plays no role in UV-induced cell cycle arrest (15).

Agents of environmental relevance but also many anti-cancer drugs introduce bulky base damage similar to UV. Checkpoint responses toward these agents prevent genetic instability, and an understanding of the molecular details involved in damage recognition will have many implications for important issues related to human health and, in particular to cancer, its prevention and its treatment.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA87381 and ES01163.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Division of Biological Sciences, University of Kansas, Haworth Hall, 1200 Sunnyside, Lawrence, KS 66045.

§ Present address: Dept. of Cell Biology and Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107. To whom correspondence should be addressed. Tel.: 817-735-2048; Fax: 817-735-2610.

Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M300061200

    ABBREVIATIONS

The abbreviations used are: PCNA, proliferating cell nuclear antigen; ATR, ATM (ataxia telangiectasia-mutated) and Rad3-related; NER, nucleotide excision repair; FACS, fluorescence-activated cell sorting.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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