The Aspergillus nidulans uvsB Gene Encodes an ATM-Related Kinase Required for Multiple Facets of the DNA Damage Response

Corresponding author: Steven D. Harris, Department of Microbiology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3205., sharris{at}nso2.uchc.edu (E-mail)

Communicating editor: R. H. DAVIS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In Aspergillus nidulans, uvsB and uvsD belong to the same epistasis group of DNA repair mutants. Recent observations suggest that these genes are likely to control cell cycle checkpoint responses to DNA damage and incomplete replication. Consistent with this notion, we show here that UVSB is a member of the conserved family of ATM-related kinases. Phenotypic characterization of uvsB mutants shows that they possess defects in additional aspects of the DNA damage response besides checkpoint control, including inhibition of septum formation, regulation of gene expression, and induced mutagenesis. The musN227 mutation partially suppresses the poor growth and DNA damage sensitivity of uvsB mutants. Although musN227 partially suppresses several uvsB defects, it does not restore checkpoint function to uvsB mutants. Notably, the failure of uvsB mutants to restrain septum formation in the presence of DNA damage is suppressed by the musN227 mutation. We propose that UVSB functions as the central regulator of the A. nidulans DNA damage response, whereas MUSN promotes recovery by modulating a subset of the response.

SUCCESSFUL cellular proliferation is absolutely dependent on replication and segregation of the genome with the highest fidelity. The DNA damage response (DDR) is a protective mechanism that ensures the maintenance of genomic integrity during cellular reproduction. The DDR consists of extensive repair systems that deal directly with DNA damage, as well as surveillance systems, known as checkpoints, which arrest the cell cycle and provide a period of time for repair before the critical events of replication and segregation occur (FRIEDBERG et al. 1995 ). Activation of the DNA damage checkpoint can halt the cell cycle at the G1-S transition (SIEDE et al. 1993 , SIEDE et al. 1994 ), slow progression through S phase (PAULOVICH and HARTWELL 1995 ), or cause arrest at the G2-M transition (WEINERT and HARTWELL 1988 ). Extensive molecular characterization of DNA damage checkpoint pathways in numerous eukaryotic organisms has revealed that regardless of the specific effect, checkpoint activation is controlled by the conserved family of ataxia telangiectasia mutated (ATM) kinases (ZAKIAN 1995 ; ROTMAN and SHILOH 1998 ). In response to the presence of DNA damage, these PI-3 related kinases modulate cell cycle progression by phosphorylating multiple substrates, including p53 (BANIN et al. 1998 ; CANMAN et al. 1998 ), and the Rad53/Cds1/Chk2 protein kinases (SANCHEZ et al. 1996 ; MATSUOKA et al. 1998 ; BROWN et al. 1999 ). In addition to their checkpoint function, several observations suggest that the ATM kinases regulate additional facets of the eukaryotic DDR. For example, in Saccharomyces cerevisiae, the ATM kinase Mec1p is required for the transcriptional induction of genes involved in the metabolism and repair of DNA damage (ABOUSSEKHRA et al. 1996 ; KISER and WEINERT 1996 ). Furthermore, in Schizosaccharomyces pombe and human cells, ATM kinases have been implicated in the control of DNA damage-induced mutagenesis (GENTNER et al. 1978 ; BENNETT and RAINBOW 1988 ; HILGERS et al. 1989 ). The functional importance of the ATM kinases in the DDR is exemplified by the observation that mutations in the human ATM gene cause the cancer-prone syndrome ataxia telangiectasia (SAVITSKY et al. 1995 ).

Extensive genetic analyses have resulted in the characterization of a large number of DNA repair mutants in the filamentous fungus Aspergillus nidulans (reviewed by KAFER and MAY 1998 ). Furthermore, a subset of these mutants has been sorted into four distinct epistasis groups (KAFER and MAYOR 1986 ; CHAE and KAFER 1993 ), and representative members from three of these groups have been cloned (YOON et al. 1995 ; KAFER and MAY 1997 ; HAN et al. 1998 ). Despite this progress, the A. nidulans DDR remains poorly characterized. Analysis of cell cycle responses to DNA damage has shown that A. nidulans possesses a G2/M checkpoint that depends on inhibitory tyrosine phosphorylation of the cyclin-dependent kinase NIMX^cdk1 (YE et al. 1997 ). We have shown that this pathway also mediates DNA damage-induced inhibition of cell division (septum formation; HARRIS and KRAUS 1998 ). We have also demonstrated that uvsB and uvsD mutants, which define the remaining uncharacterized epistasis group of repair mutants, are unable to restrain septum formation in the presence of DNA damage (HARRIS and KRAUS 1998 ). This result, together with the observation that uvsB and uvsD mutants are sensitive to a diverse array of DNA damaging agents (KAFER and MAYOR 1986 ; HARRIS and KRAUS 1998 ), suggests that these genes encode proteins involved in DNA damage checkpoint function. Here, we demonstrate that UVSB is a member of the family of ATM kinases that have been implicated in the cellular response to DNA damage. We show that UVSB is required for multiple facets of the A. nidulans DDR, including nuclear division arrest, induced mutagenesis, and possibly, DNA damage-regulated gene expression. Furthermore, we report that the musN227 mutation partially suppresses specific DDR defects caused by uvsB mutations. We suggest that UVSB functions as the central regulator of the A. nidulans DDR, whereas MUSN is required for recovery and the resumption of normal cellular proliferation.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, media, and growth conditions:
The following strains were used in this study: A28 (pabaA6 biA1), A781 (nimA5; wA2), A826 (suA1adE20 adE20 biA1; uvsB505 ssbA1; sB3; choA1; chaA1), AAH1 (uvsB110; nimA5), AAH2 (uvsB110; musN227; nimA5), AAH4 (uvsD153; musN227; wA2), AAH11 (uvsB505; musN227; chaA1), AAS280 (musN227; nimX^cdc2AF;wA2), AAS315 (uvsB110; musN227 pabaA1 acrA1 actA1 riboB2 chaA1), AAS381 (sepB3; uvsB110; musN227; chaA1), ASH201 (uvsB110; chaA1), ASH202 (sepB3; uvsB110; chaA1), ASH204 (uvsB110; argB2; wA2), ASH206 (uvsD153; wA2), ASH208 (sepB3; pabaA6; wA2), ASH286 (nimX^cdc2AF; wA2), ASH380 (sepB3; musN227; wA2), and ASH383 (musN227; chaA1).

Media used were MAG (2% dextrose, 2% malt extract, 0.2% peptone, trace elements, and vitamins; pH 6.5), CM (1% dextrose, 0.2% peptone, 0.1% yeast extract, 0.1% casamino acids, nitrate salts, trace elements, and vitamins; pH 6.5), MNV (1% dextrose, nitrate salts, trace elements, and vitamins; pH 6.5), or YGV (2% dextrose, 0.5% yeast extract, and vitamins). Trace elements, nitrate salts, and vitamins were as described previously (KAFER 1977 ). Uridine (250 mM) and uracil (500 mM) were added as necessary to support the growth of pyrimidine requiring strains. Triton X-100 (0.01%) was added to solid media to promote colonial growth for viability assays. Hydroxyurea (HU; Sigma Chemical Co., St. Louis), diepoxyoctane (DEO; Aldrich Chemical Co., Milwaukee) and methyl methane sulfonate (MMS; Aldrich Chemical Co.) were added to the appropriate concentration after autoclaving. 5-Fluoroorotic acid (5-FOA; Angus Buffers and Biochemicals, Niagara Falls, NY) was added to media at a concentration of 1 mg/ml after autoclaving. All genetic manipulations performed in this study have been described previously (HARRIS et al. 1994 ). sepB3; uvsB110; musN227 triple mutants were identified as temperature-sensitive segregants that were sensitive to 5 mM HU but exhibited slightly better growth on 0.005 and 0.01% MMS than the sepB3;uvsB110 double mutant. The musN227; nimX^cdc2AF double mutants were identified as segregants that were sensitive to 5 mM HU and exhibited enhanced sensitivity to MMS as compared to the musN227 mutant. Double and triple mutant genotypes were confirmed by a backcross to wild type.

Cloning of the uvsB gene:
Strain ASH204 was cotransformed with the argB-based autonomously replicating plasmid pDHG25 (GEMS et al. 1991 ) and pools of cosmids from a chromosome IV specific cosmid library (BRODY et al. 1991 ). Transformants that could grow on MNV media containing 5 mM HU were recovered at 32°. Pools were subdivided until a single cosmid (W24C04) that could complement the HU hypersensitivity of uvsB110 mutants was identified. Subsequent transformations with fragments derived from this cosmid localized the complementing activity to a 10-kb EcoRI-NotI fragment. Sequencing of this fragment and primer synthesis were performed by the Molecular Core Facility at the University of Connecticut Health Center. The BLAST algorithm at the NCBI (ALTSCHUL et al. 1997 ) was used to search the available DNA sequence databases. Several approaches were used to locate introns in the uvsB sequence, including (i) alignment of the amino acid sequence with that of its closest orthologue Rad3p, (ii) examination of the sequence for the presence of conserved splicing sequences (GURR et al. 1987 ), and (iii) reverse transcriptase (RT)-PCR. RT-PCR was performed using the RETROscript First Strand Synthesis kit (Ambion, Austin, TX).

To replace the uvsB gene with the argB marker, plasmid pAS1 was constructed. First, plasmid pASUB10, which contains the entire uvsB open reading frame, was digested to completion with BamHI. The resulting 6.5-kb BamHI fragment containing the vector backbone plus flanking uvsB sequences was then ligated to a 1.8-kb BamHI fragment containing argB.

Viability assays:
Conidiospores from the appropriate strains were diluted and plated at ~100 conidia per plate on CM + Triton X-100 media containing the appropriate concentration of HU or MMS. For UV viability assays, conidiospores were plated and allowed to germinate for 4.5 hr before UV irradiation. Plates were incubated for 3 days at 32° and the number of survivors on each plate counted. For each treatment, duplicate platings were performed and the average number of survivors calculated. Viability was determined as the percentage of colonies on treated plates compared to untreated controls.

Mutagenesis assays:
For each strain tested, 10⁷ conidiospores were treated for 30 min at 30° in 1 ml YGV containing 0.05% MMS. The MMS was subsequently neutralized with sodium thiosulfate and the conidiospores were washed with sterile distilled water. Spores were diluted and plated onto MNV + 5-FOA (to select pyrimidine requiring mutants) and CM + Triton X-100 (to measure viability). Plates were incubated at 32° for 3 days. All platings were performed in quadruplicate, and the mean number of survivors and 5-FOA-resistant colonies determined. The level of mutagenesis was expressed as the number of FOA-resistant colonies per 10⁶ viable conidia.

Microscopy:
Cells were grown, fixed, and stained as previously described (HARRIS et al. 1994 ; HARRIS and KRAUS 1998 ). Hoechst 33258 and Calcofluor were used to visualize nuclei and septa, respectively. Nuclear division kinetics and septation indices were determined as previously described (HARRIS et al. 1994 ; HARRIS and KRAUS 1998 ). To assess the effect of the uvsB110 and musN227 mutations on mitotic entry in synchronized germlings, strains containing the temperature-sensitive nimA5 mutation were exposed to 0.025% MMS following a 6-hr incubation at 43.5°. The extent of mitotic entry was determined by measuring the chromosome mitotic index (HARRIS and HAMER 1995 ; n = 100).

Isolation of total RNA and Northern analysis:
Strains were grown in liquid YGV for 12 hr at 28° with constant shaking. MMS was added to a concentration of 0.025% and the incubation continued for a further 3 hr before harvesting. For sepB3 strains, cultures were grown for 8 hr at 28° before shifting to 42° and incubating for an additional 8 hr before harvesting. Isolation of total RNA was performed as described (DOBINSON et al. 1993 ).

RNA gels were run, blotted, and probed according to standard protocols (AUSUBEL et al. 1995 ). The uvsC probe consisted of a 0.6-kb SphI cDNA fragment derived from plasmid pGW1525 (VAN HEEMST et al. 1997 ). The actin probe used as a loading control was a 5.1-kb HindIII genomic fragment from plasmid pSH16. Blots were exposed to Kodak X-OMAT AR film and an intensifying screen for 24–48 hr at -70°. Band intensity was quantitated by phosphoimager analysis.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cloning of the uvsB gene:
The uvsB gene had previously been mapped to chromosome IV (JANSEN 1970 ). By transforming pools of cosmids from chromosome IV into a uvsB110 mutant, we identified a single cosmid, W24C04, that was capable of complementing the HU sensitivity caused by the uvsB110 mutation. Additional transformation experiments using fragments derived from W24C04 identified a 10-kb EcoRI-NotI fragment that possessed complementing activity. The complete DNA sequence of both strands of this fragment was determined. Analysis of the sequence revealed that uvsB encodes a predicted protein of 2454 amino acids, with an estimated molecular weight of 277 kD. Searches of available databases revealed that UVSB belongs to the ATM family of PI-3 related kinases (ZAKIAN 1995 ). In particular, UVSB is most similar to S. pombe Rad3p, S. cerevisiae Mec1p, Drosophila melanogaster MEI-41, and human ATM and RAD3-related protein (ATR) (JEGGO et al. 1998 ; Fig 1). The identity with its closest orthologue, Rad3p, is primarily confined to three regions (Fig 1): the C-terminal PI-3 kinase domain (55%), the Rad3 domain (28%), and the putative leucine zipper region (30%).

View larger version (20K): In this window In a new window Download PPT slide   Figure 1. Molecular characterization of UVSB. A schematic representation of UVSB is shown, illustrating the locations of the PI-3 kinase domain, the RAD3 domain, and the putative leucine zipper. Levels of sequence identity between UVSB and other ATM-related kinases within each domain are also provided. Percentage identity was calculated by the BLAST algorithm at NCBI. The aminoterminal 647 amino acids show no significant similarity to any sequence in the database. The GenBank accession number for the uvsB sequence is AF176575. The solid black line depicts the region of UVSB missing in the deletion plasmid pAS1.

To determine if uvsB is an essential gene, a plasmid was constructed in which 75% of the uvsB open reading frame was replaced with the selectable argB marker (Fig 1). When transformed into a haploid strain, we failed to recover Arg⁺ spores displaying phenotypes consistent with loss of UVSB function (i.e., HU or MMS sensitivity, slow growth; A. HOFMANN and S. HARRIS, unpublished results). Furthermore, DNA blot analysis of a set of Arg⁺ transformants displaying wild-type phenotypes revealed that the plasmid had integrated ectopically in all cases. These observations imply that the uvsB gene is essential. Moreover, they also suggest that the existing uvsB alleles are hypomorphic.

UVSB is required for the DNA damage checkpoint:
Mutations in uvsB cause sensitivity to UV irradiation, DNA alkylating agents, and the replication inhibitor HU (JANSEN 1970 ; HARRIS and KRAUS 1998 ). Recently, it became apparent that UVSB was likely to function as a component of the DNA damage checkpoint, as mutations in uvsB abolish the cell cycle delay and inhibition of septum formation triggered by the exposure of predivisional hyphae to DNA damaging agents (HARRIS and KRAUS 1998 ).

To confirm the role of UVSB in the DNA damage checkpoint, we examined the kinetics of nuclear division in a sepB3 uvsB110 double mutant. SEPB is likely to function in chromosomal DNA metabolism, since temperature-sensitive sepB mutations cause increased levels of mitotic recombination and chromosome nondisjunction (HARRIS and HAMER 1995 ). Consistent with previous observations, sepB3 mutants displayed a pronounced nuclear division delay at restrictive temperature (HARRIS and HAMER 1995 ; Fig 2). This delay is completely abolished by the uvsB110 mutation, as sepB3 uvsB110 double mutants undergo nuclear division with kinetics similar to wild type (Fig 2). We have also shown that the uvsB110 mutation can abrogate the mitotic delay observed when cells synchronized at the G2-M transition using the nimA5 mutation are released into media containing 0.025% MMS. In particular, following 20-min exposure to MMS at 42°, 49% of nimA5 uvsB110 germlings had entered mitosis compared to only 8% of nimA5 germlings. The fraction of untreated germlings that had entered mitosis was 58 and 36% for nimA5 uvsB110 and nimA5 germlings, respectively. These observations demonstrate that UVSB is required for the damage-induced inhibition of cell cycle progression.

View larger version (18K): In this window In a new window Download PPT slide   Figure 2. UVSB is required for the nuclear division delay triggered by the sepB3 mutation. Conidia from strains A28 (wild type, circles), ASH208 (sepB3, squares), ASH202 (sepB3 uvsB110, triangles), ASH380 (sepB3musN227, diamonds), and AAS381 (sepB3uvsB110musN227, inverted triangles) were germinated on coverslips for 3 hr at 42°. Thereafter, one coverslip was removed every 30 min over a 5-hr period and processed for microscopy. Coverslips were stained with Hoechst 33258 and Calcofluor to visualize nuclei and septa, respectively. For each strain and time point, 200 randomly selected germlings were scored for the number of nuclei present. Data points represent the percentage of germlings possessing four or more nuclei.

UVSB is required for additional aspects of the DDR:
Phenotypic characterization of uvsB mutants suggested that UVSB may have additional roles in the DDR other than simply controlling the checkpoint response. For example, uvsB110 mutants are much more sensitive to MMS treatment than nimX^cdc2AF mutants, which appear to be solely defective in checkpoint function (Fig 3; YE et al. 1997 ). In addition, uvsB mutations cause mitotically quiescent conidia, in which checkpoint function is presumably dispensable, to be sensitive to both UV irradiation and MMS (KAFER and MAYOR 1986 ).

View larger version (16K): In this window In a new window Download PPT slide   Figure 3. uvsB110 mutants are more sensitive to MMS than nimXcdc2AF mutants. Conidia from strains A28 (wild type, circles), ASH201 (uvsB110, triangles), and ASH286 (nimXcdc2AF, diamonds) were diluted and plated at ~100 conidia per plate on CM + Triton X-100 media containing the indicated concentration of MMS. The number of survivors on each plate was determined after 3 days incubation at 32°. The percentage of viable colonies represents the percentage of colonies remaining on the treated plates as compared to the untreated control plates. The data plotted represent the average of four plates tested per dose of MMS.

These observations led us to examine other aspects of the DDR that may depend upon UVSB function. One critical element of the DDR is the induction of mutagenesis (FRIEDBERG et al. 1995 ). Using 5-FOA resistance as an assay, the uvsB110 mutation virtually abolished induced mutagenesis (Table 1). Notably, a similar phenotype has also been observed in AT cells (BENNETT and RAINBOW 1988 ; HILGERS et al. 1989 )

Another critical element of the DDR is the transcriptional induction of genes involved in the metabolism and repair of DNA damage (WITKIN 1991 ; ABOUSSEKHRA et al. 1996 ; KISER and WEINERT 1996 ) In S. cerevisiae, the transcriptional response to DNA damage has been shown to be dependent on Mec1p function (KISER and WEINERT 1996 ). Similarly, in humans, ATM likely triggers significant changes in gene expression through its ability to phosphorylate p53 (BANIN et al. 1998 ; CANMAN et al. 1998 ). Given the similarity between UVSB and both Mec1p and ATM, it seemed reasonable to test whether UVSB is similarly required for the transcriptional response to DNA damage in A. nidulans. One A. nidulans gene that has been shown to be induced in the presence of DNA damage is uvsC (VAN HEEMST et al. 1997 ), which encodes the A. nidulans orthologue of S. cerevisiae Rad51p. Treatment of wild-type cells with MMS results in a 3.3-fold increase in uvsC transcript levels (Table 2). This increase is not due to artificial cell cycle synchronization caused by MMS treatment, since it still occurred in hyphae arrested at the G2/M transition by the nimA5 mutation. For example, in one experiment, nimA5 mutants arrested at 43.5° and treated with 0.025% MMS for 3 hr showed a 5.8-fold induction of uvsC transcript. The elevation of uvsC transcript levels caused by MMS treatment is reduced by ~35% in a uvsB110 mutant (Table 2). As an independent means of testing the role of UVSB in the transcriptional response to DNA damage, we examined uvsC transcript levels in sepB3 mutants that had been shifted to restrictive temperature. The observation that these conditions cause an increase in uvsC transcript levels suggests that the sepB3 mutation generates a DNA damage signal (Table 3). However, as with MMS-treated wild-type cells, this response is only partially diminished (~50%) by the uvsB110 mutation (Table 3). Thus, given the relatively weak effect of the uvsB110 mutation on uvsC expression, we can only conclude at this time that UVSB plays a modest role at best in the transcriptional response to DNA damage in A. nidulans.

The musN227 mutation suppresses specific DDR defects caused by uvsB mutations:
The recessive musN227 mutation was originally isolated in a screen for MMS-sensitive mutants (KAFER and MAYOR 1986 ). Construction of double mutants for epistasis analysis revealed that the musN227 mutation could partially suppress the poor growth and DNA damage sensitivity of several different uvsB mutant alleles (KAFER and CHAE 1994 ). Consistent with this result, we found that the musN227 mutation caused a 10-fold increase in the viability of uvsB110 and uvsB505 mutants exposed to low doses of MMS (Fig 4). A similar effect was noted at low doses of HU (A. HOFMANN and S. HARRIS, unpublished results). The partial suppression conferred by the musN227 mutation was not limited to uvsB mutations, since musN227 uvsD153 double mutants also grew better at low doses of MMS (Fig 4).

View larger version (21K): In this window In a new window Download PPT slide   Figure 4. The musN227 mutation partially suppresses the MMS sensitivity of uvsB and uvsD mutants. Conidia from strains A28 (wild type, open circles), ASH201 (uvsB110, open triangles), A826 (uvsB505, open squares), ASH206 (uvsD153, open diamond), ASH383 (musN227, inverted open triangle), AAS315 (uvsB110 musN227, solid triangles), AAH11 (uvsB505 musN227, solid squares), and AAH4 (uvsD153 musN227, solid diamonds) were diluted and plated at ~100 conidia per plate on CM + Triton X-100 media containing the indicated concentration of MMS. The number of survivors on each plate was scored after 3 days incubation at 32°. The percentage of viable colonies represents the percentage of colonies remaining on the treated plates as compared to the untreated control plates. Data represent the average of four plates counted for each dose.

Since UVSB appears to be required for multiple aspects of the DDR, we sought to determine which elements of the DDR are affected by the musN227 mutation. We first assessed the ability of the musN227 mutation to restore damage-induced nuclear division delay to uvsB110 mutants. To do this, we constructed a sepB3 uvsB110 musN227 triple mutant and examined the kinetics of nuclear division at the restrictive temperature. The triple mutant divided with kinetics similar to that of the sepB3 uvsB110 double mutant (Fig 2), indicating that the musN227 mutation did not restore the nuclear division delay. The observation that the mitotic delay is not restored in nimA5 uvsB110 musN227 triple mutants that have been synchronized at G2-M and then released into MMS supports this notion. In particular, following 20-min exposure to 0.025% MMS at 42°, 35% of nimA5 uvsB110 musN227 germlings had entered mitosis, compared to 49% of nimA5 uvsB110 germlings and 8% of nimA5 germlings. These results suggest that the musN227 mutation must affect other UVSB-dependent aspects of the DDR.

As previously shown, the uvsB110 mutation abolishes the damage-induced inhibition of septum formation (HARRIS and KRAUS 1998 ; Table 4). To determine the effect of the musN227 mutation on this aspect of the DDR, we assessed the ability of the sepB3 uvsB110 musN227 triple mutant to undergo septation at the restrictive temperature. Remarkably, the level of septation was severely reduced by the addition of the musN227 mutation to the sepB3uvsB110 background (Table 4). Thus, we conclude that musN227 partially restores to uvsB mutants the ability to block septation in the presence of DNA damage.

The uvsB110 mutation causes a defect in induced mutagenesis. To determine the effect of the musN227 mutation on this aspect of the DDR, we tested the ability of musN227 uvsB110 double mutants to form FOA-resistant colonies following treatment with MMS. The musN227 mutation partially restored induced mutagenesis to the uvsB110 mutant (Table 1). Indeed, the musN227 mutation itself appears to cause a slight induced mutator phenotype (Table 1). Thus, we conclude that musN227 partially suppresses the inability of uvsB mutants to induce mutagenesis when exposed to a DNA damaging agent.

The uvsB110 mutation causes a modest reduction of uvsC transcript levels in the presence of DNA damage. To assess the effect of the musN227 mutation on this aspect of the uvsB phenotype, we examined transcript levels from uvsC in musN227 uvsB110 double mutants treated with MMS. The observation that the double mutant only displayed a slight increase in uvsC transcript levels suggests that the musN227 mutation does not significantly restore DNA damage-induced transcription in uvsB mutants (Table 2).

The effect of musN227 on septation is independent of NIMX^cdk1:
In A. nidulans, the presence of DNA damage triggers inhibitory Tyr-15 phosphorylation of the cdk NIMX^cdk1 (YE et al. 1997 ). This phosphorylation event leaves NIMX^cdk1 in an inactive state, resulting in inhibition of mitosis and septum formation (YE et al. 1997 ; HARRIS and KRAUS 1998 ). The nimX^cdc2AF mutation makes NIMX^cdk1 refractory to Tyr-15 phosphorylation (YE et al. 1996 ). As a result, nimX^cdc2AF mutants septate at high levels despite the presence of DNA damage (HARRIS and KRAUS 1998 ). If the musN227 mutation affects the DNA damage-induced inhibition of septum formation by regulating Tyr-15 phosphorylation of NIMX^cdk1, it should have no effect on septation in nimX^cdc2AF mutants. To test this possibility, musN227 nimX^cdc2AF double mutants were constructed and tested for the ability to septate in the presence of the alkylating agent DEO. The musN227 mutation decreased the level of septation observed in the nimX^cdc2AF mutant (Table 5). This observation suggests that MUSN regulates septation via a separate pathway from that which regulates NIMX^cdk1 tyrosine phosphorylation. To further explore this possibility, we compared the MMS sensitivity of the musN227 nimX^cdc2AF double mutant to the parental single mutants. The double mutant showed enhanced sensitivity to MMS (Fig 5), indicating that musN227 and nimX^cdc2AF belong to different epistasis groups of DNA repair mutants.

View larger version (19K): In this window In a new window Download PPT slide   Figure 5. The musN227 and nimXcdc2AF mutations belong to different epistasis groups. Conidia from strains A28 (wild type, circles), ASH383 (musN227, squares), ASH286 (nimXcdc2AF, triangles), and AAS280 (musN227 nimXcdc2AF, diamonds) were diluted and plated at ~100 conidia per plate on CM + Triton X-100 media containing the indicated concentration of MMS. The number of survivors on each plate was scored after 3 days incubation at 32°. The percentage of viable colonies represents the percentage of colonies remaining on the treated plates as compared to the untreated control plates. Data represent the average of six plates counted for each dose.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mutations in the uvsB and uvsD genes define one of four epistatic groups of DNA repair mutants in A. nidulans (KAFER and MAYOR 1986 ). Characterization of the phenotypes caused by these mutations suggested that uvsB and uvsD may encode proteins required for both the DNA damage and the DNA replication checkpoints (YE et al. 1997 ; HARRIS and KRAUS 1998 ). Here, the function of UVSB in the A. nidulans DNA damage response is described. In particular, we show that (i) UVSB is a member of the conserved family of ATM-related protein kinases that control checkpoint signal transduction, and (ii) UVSB is required for several aspects of the DDR, including nuclear division arrest, induced mutagenesis, and perhaps, DNA damage-induced gene expression. In addition, we demonstrate that a subset of uvsB defects can be partially suppressed by the musN227 mutation. We propose that UVSB functions as the central regulator of the A. nidulans DDR, whereas MUSN promotes recovery and the resumption of normal proliferative growth.

UVSB is a member of the ATM-related family of protein kinases:
DNA sequence analysis shows that UVSB is a member of the ATM-related protein kinase family. In particular, UVSB shares greatest similarity with the ATM subfamily that includes human ATR, S. cerevisiae Mec1p, and S. pombe Rad3p. The similarity extends beyond the conserved carboxy-terminal PI-3 kinase domain and includes both the Rad3 domain and a putative leucine zipper (JEGGO et al. 1998 ). Despite the presence of the PI-3 kinase domain, members of the ATM family display protein kinase activity (BENTLEY et al. 1996 ; BANIN et al. 1998 ; CANMAN et al. 1998 ; CLIBY et al. 1998 ). Furthermore, this activity underlies the ability of the ATM kinases to trigger a cell cycle checkpoint response. For example, ATM, Mec1p, and Rad3 each appear to activate checkpoint responses by phosphorylating downstream protein kinases (i.e., Rad53/Cds1/Chk2 and Chk1p; SANCHEZ et al. 1996 ; MARTINHO et al. 1998 ; MATSUOKA et al. 1998 ; BROWN et al. 1999 ). The presence of the conserved PI-3 kinase domain in UVSB suggests that it also exerts its checkpoint effect by phosphorylating downstream effector proteins. Three observations suggest that UVSD may be such a target. First, uvsB and uvsD mutations cause a similar spectrum of phenotypes (HARRIS and KRAUS 1998 ; KAFER and MAY 1998 ), including failure to activate cell cycle checkpoints in the presence of DNA damage. Second, the epistatic relationship between uvsB and uvsD mutations demonstrates that both genes function in the same pathway (KAFER and MAYOR 1986 ). Third, UVSD presumably functions downstream of UVSB, since preliminary experiments show that unlike uvsB mutants, the uvsD153 mutation does not affect DNA damage-induced gene expression (A. HOFMANN and S. HARRIS, unpublished results).

UVSB is required for multiple facets of the A. nidulans DDR:
Since the DNA damage sensitivity caused by uvsB mutations greatly exceeds that caused by a typical DNA damage checkpoint mutant (i.e., nimX^cdc2AF; YE et al. 1997 ), we presumed that UVSB is required for other facets of the A. nidulans DDR in addition to the checkpoint response. In accordance with this hypothesis, we have previously shown that UVSB is required for the DNA damage-induced inhibition of septum formation (HARRIS and KRAUS 1998 ). Results presented here suggest that UVSB could possibly play a role in DNA damage-induced gene expression. Transcriptional induction of at least one DNA damage-responsive gene was found to be slightly affected by the uvsB110 mutation. Although we cannot eliminate the possibility that DNA damage affects uvsC transcript stability, the presence of conserved MluI cell cycle box (MCB) elements (MCINTOSH 1993 ) in the uvsC promoter (VAN HEEMST et al. 1997 ) suggests that, as in S. cerevisiae and S. pombe (HARRIS et al. 1996 ; BACHANT and ELLEDGE 1998 ), the effect is probably exerted at the level of transcriptional induction. Since the uvsB110 mutation is likely to be hypomorphic, and its effect on uvsC transcript levels was rather small, analysis of DNA damage-induced expression of uvsC in a uvsB null background should provide further insight into the role of UVSB in this facet of the DDR. In addition, the expression of other genes showing comparable, if not greater, levels of DNA damage induction should be examined.

Another important aspect of the DDR is the induction of mutagenic DNA repair (FRIEDBERG et al. 1995 ). Using a forward mutagenesis assay, we have found that a uvsB mutation virtually abolishes induced mutagenesis in A. nidulans. Other ATM family members have also been implicated in the control of mutagenesis. For example, S. pombe rad3 mutants display pronounced defects in forward mutation (GENTNER et al. 1978 ). In addition, examination of viral mutagenesis in infected AT cells demonstrated that they were defective for an inducible error-prone repair process (BENNETT and RAINBOW 1988 ; HILGERS et al. 1989 ). The mechanism by which ATM-related protein kinases influence DNA damage-induced mutagenesis is currently unknown. However, it is not inconceivable that the DNA damage-induced expression of genes required for mutagenic DNA repair is regulated by ATM family members.

Multiple defects observed in uvsB mutants are suppressed by the musN277 mutation:
The musN227 mutation was originally isolated in a screen for mutants sensitive to MMS (KAFER and MAYOR 1986 ). During the construction of double mutants for epistasis analysis, it was noted that musN227 partially suppressed the slow growth and DNA damage sensitivity of uvsB mutants (KAFER and CHAE 1994 ). We have confirmed this observation and, furthermore, shown that musN227 modifies several, but not all, of the phenotypes caused by uvsB mutations. In particular, the inability of uvsB110 mutants to restrain septum formation and induce mutagenesis in the presence of DNA damage was negated by the musN227 mutation. In contrast, musN227 does not appear to affect the loss of DNA damage-induced cell cycle delay caused by uvsB mutations, and its effect on uvsC transcription was not significant. The retention of the checkpoint defect in the musN227 uvsB110 double mutant may account, in part, for the partial nature of the suppression. Collectively, our observations suggest that the musN227 mutation does not cause a general increase in UVSB activity. Instead, we propose that musN227 modifies a specific UVSB function that has gone awry in uvsB mutants. Furthermore, we suggest that this function may be the ability of UVSB to properly modulate the activity of gene products responsible for inhibiting septation and inducing mutagenesis in response to the presence of DNA damage.

Several observations suggest that the suppression of uvsB defects by the musN227 mutation does not reflect a direct physical interaction between MUSN and UVSB. First, musN227 can suppress defects caused by different alleles of uvsB (Fig 4). Second, the musN227 mutation also suppresses DDR-related defects caused by the uvsD153 mutation (KAFER and CHAE 1994 ; Fig 4). Third, musP234, a mutant recovered from the same screen as musN227, suppresses uvsB defects to the same apparent extent as the musN227 mutation (KAFER and CHAE 1994 ; A. HOFMANN and S. HARRIS, unpublished observations). We propose that the musN and musP mutations affect a regulatory pathway that normally antagonizes UVSB function. For example, MUSN and MUSP may be components of a pathway that downregulates the DNA damage-induced transcriptional response to promote recovery from the DDR and the resumption of normal proliferation. In the mus uvsB double mutants, the crippled DDR caused by the uvsB mutation may be sufficiently enhanced by the mus mutation so that some degree of resistance to DNA damage is restored. An important, but as yet untested, prediction of this model is that the mus mutations may have no effect if the DDR is completely abolished (i.e., in a uvsB null mutant).

Regulation of septum formation by DNA damage:
We have previously shown that the presence of DNA damage inhibits septum formation in A. nidulans (HARRIS and KRAUS 1998 ). Furthermore, we proposed that DNA damage triggers a regulatory response that leads to a reduction in the activity of the cyclin-dependent kinase NIMX^cdk1 below a threshold needed to trigger septation. Our genetic analyses indicated that the ultimate effect of this response is to increase the level of inhibitory NIMX^cdk1 tyrosine phosphorylation (HARRIS and KRAUS 1998 ). However, two lines of evidence suggest that DNA damage may regulate septum formation via an additional mechanism. First, when incubated at restrictive temperature, the level of septation in sepB3 uvsB double mutants is consistently higher than that seen in sepB3 nimX^cdc2AF mutants (HARRIS and KRAUS 1998 ). If the sole effect of DNA damage on septation was mediated by inhibitory tyrosine phosphorylation of NIMX^cdk1, the level of septation in the double mutants should be similar. Second, the observation that the musN227 mutation affects septation independent of NIMX^cdk1 tyrosine phosphorylation implies that a second pathway for DNA damage regulation of septation exists. We propose that this pathway is controlled by UVSB. Moreover, since NIMX^cdk1 is required for septum formation (HARRIS and KRAUS 1998 ), we suggest that this pathway affects septation by negatively regulating NIMX^cdk1 activity through a mechanism other than tyrosine phosphorylation. For example, UVSB, and potentially MUSN, could modulate the expression of a cyclin(s) that associates with NIMX^cdk1 to control septation (Fig 6). Although this cyclin could be NIME (O'CONNELL et al. 1992 ; HARRIS and KRAUS 1998 ), it remains possible that A. nidulans possesses an additional cyclin whose accumulation is delayed by the presence of DNA damage (i.e., similar to S. cerevisiae Cln1p and Cln2p; SIDOROVA and BREEDEN 1997 ). We note that the ability of DNA damage to specifically block cytokinesis in human cells may rely upon a similar ATM-dependent mechanism (HERMEKING et al. 1997 ; BUNZ et al. 1998 ).

View larger version (14K): In this window In a new window Download PPT slide   Figure 6. A tentative model for the regulation of septum formation by DNA damage in A. nidulans. In the presence of DNA damage, activated UVSB is proposed to modulate NIMXcdk1 activity via two mechanisms: inhibition of Tyr-15 dephosphorylation and inhibition of cyclin accumulation. Septum formation is blocked by reduced NIMXcdk1 activity. See text for further details.

	ACKNOWLEDGMENTS

We thank Etta Kafer, Diane van Heemst, Xiang Ye, and Stephen Osmani for graciously providing strains or plasmids. We also thank Tom Wolkow for comments that improved the manuscript This work was supported by awards from the National Science Foundation (MCB-9513489) and the University of Connecticut Health Center Research Advisory Council.

Manuscript received August 23, 1999; Accepted for publication December 3, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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