NORF5/HUG1 is a component of the MEC1-mediated checkpoint response to DNA damage and replication arrest in Saccharomyces cerevisiae

Abstract

Analysis of global gene expression in Saccharomyces cerevisiae by the serial analysis of gene expression technique has permitted the identification of at least 302 previously unidentified transcripts from nonannotated open reading frames (NORFs). Transcription of one of these, NORF5/HUG1 (hydroxyurea and UV and gamma radiation induced), is induced by DNA damage, and this induction requires MEC1, a homolog of the ataxia telangiectasia mutated (ATM) gene. DNA damage-specific induction of HUG1, which is independent of the cell cycle stage, is due to the alleviation of repression by the Crt1p-Ssn6p-Tup1p complex. Overexpression of HUG1 is lethal in combination with a mec1 mutation in the presence of DNA damage or replication arrest, whereas a deletion of HUG1 rescues the lethality due to a mec1 null allele. HUG1 is the first example of a NORF with important biological functional properties and defines a novel component of the MEC1 checkpoint pathway.

FIG. 1

Transcription of NORF5/HUG1 is induced by replication arrest and DNA damage. (A) NORF5 transcription is upregulated in cells arrested with HU. Results are from Northern blot analysis with wild-type cells (YPH499) grown logarithmically (lane 1) and arrested with HU (lane 2) or nocodazole (Noc) (lane 3). The expression pattern observed by SAGE is indicated at the bottom (0:49:0) (40). (B) NORF5 is translated in cells arrested with HU. Western blot analysis was done with protein extracts from transformants (YMB711) containing pMB366 (3HA-NORF5 LEU2 CEN) or plasmid pMB363 (NORF5 LEU2 CEN) grown logarithmically (lanes 1 to 4) or arrested with HU (lanes 5 to 8) and probed with HA antibody as described previously. (C) Transcription of NORF5/HUG1 is HU and UV and gamma radiation induced. Results are from Northern blot analysis with wild-type cells (YPH499) grown logarithmically (lanes 1, 3, and 5), arrested with HU (lane 2), exposed to UV radiation (lane 4), or exposed to gamma radiation (lane 6). (D) HUG1 transcription is delayed upon replication arrest with HU. Results are from Northern blot analysis using logarithmically grown wild-type cells (YPH499) (lane 1) or after addition of HU (0.1 M) and incubation for 0 h (lane 2), 0.5 h (lane 3), 1.0 h (lane 4), 1.5 h (lane 5), 2.5 h (lane 6), or 3.5 h (lane 7) at 30°C. The levels of HUG1 in lanes 1 and 2 were below the background level and hence are denoted as ND (not detected). HUG1/TUB2 indicates the ratio of the intensity of the HUG1 signal to the TUB2 signal normalized to the HUG1/TUB2 ratio in lane 3 (0.5 h) set to 1.0 as described in Materials and Methods. (E) HUG1 transcription is independent of the cell cycle stage. Northern blot analysis was done with wild-type cells (YPH499) grown logarithmically (lanes 1 and 2), arrested in G₁ phase by treatment with alpha factor (lanes 3 and 4), and arrested in G₂/M with nocodazole (lanes 5 and 6), either before (lanes 1, 3, and 5) or after exposure to gamma radiation (lanes 2, 4, and 6). The arresting agents were present throughout the incubations. HUG1/TUB2 for lanes 2, 4, and 6 indicates the ratio of the intensity of the HUG1 signal to the TUB2 signal normalized to the HUG1/TUB2 ratio in control lanes 1, 3, and 5, respectively, as described in Materials and Methods.

FIG. 2

Crt1p, Ssn6p, and Tup1p are negative regulators of HUG1 transcription in the absence of DNA damage or replication arrest. (A) The promoter of HUG1 contains X-box-related sequences Xs and Xw, with strong and weak homology, respectively, to the consensus sequence in mammalian MHC class II and S. cerevisiae RNR and CRT1 genes (16, 26, 27). (B) Transcription of HUG1 in the absence of DNA damage is repressed by the Crt1p-Ssn6p-Tup1p complex. Northern blot analysis was performed with the wild-type strain (Y300) and the crt1-Δ1::LEU2 (Y577), crt4-2/tup1 (Y217), and crt8-91/ssn6 (Y231) strains, grown logarithmically (lanes 1, 3, 5, and 7) or arrested with HU (lanes 2, 4, 6, and 8). HUG1/TUB2 for lanes 2 to 8 indicates the ratio of the intensity of the HUG1 signal to the TUB2 signal normalized to the HUG1/TUB2 ratio in control lane 1 as described in Materials and Methods.

FIG. 3

Genes in the MEC1 checkpoint pathway are required for the DNA damage- and replication arrest-induced transcription of HUG1. (A) Northern blot analysis was done with logarithmically grown, HU-arrested, UV or gamma radiation-exposed cells. The strains used were isogenic to the wild-type strain (W1588-3A) and the sml1Δ::HIS3 (U952-3C) and mec1Δ::TRP1 sml1Δ::HIS3 (U953-61D) strains. HUG1/TUB2 indicates the ratio of the intensity of the HUG1 signal to the TUB2 signal normalized to the HUG1/TUB2 ratio in control lanes 1, 2, and 3 (lanes 4, 7, and 10 normalized to lane 1, lanes 5, 8, and 11 to lane 2, and lanes 6, 9, and 12 to lane 3) as described in Materials and Methods (B, C and D) Northern blot analysis was done with strains grown logarithmically, arrested with HU (B), or exposed to UV (C) or gamma (D) radiation. The strains used were wild type (TWY397), rad53/mec2-1 (TWY312), mec3-1 (TWY316), wild type (Y203), and dun1 (Y203-dun1). Two lanes between lanes 2 and 3 in panels B, C, and D were deleted because they represented data not relevant to the experiment. HUG1/TUB2 indicates the ratio of the intensity of the HUG1 signal to the TUB2 signal in cells treated with HU or UV or gamma radiation and normalized to the HUG1/TUB2 ratio in control lanes without treatment (lane 1 normalized to lane 2, lane 3 to lane 4, lane 5 to lane 6, lane 7 to lane 8, lane 9 to lane 10, and lane 11 to lane 12). Transcription of TUB2 is not induced by UV or gamma radiation; the data reflect unequal loading of the lanes as evidenced by ethidium bromide staining of the gels (data not shown). (For Fig. 2D, lane 5, the level of HUG1 was below the background level and hence was denoted as not detected [ND].) The wild-type strain isogenic to the rad53 and mec3 mutants is represented in lanes 1 and 2. The wild-type strain isogenic to the dun1 mutant is represented in lanes 9 and 10. (E) Northern blot analysis using logarithmically grown cells, arrested with HU or exposed to gamma radiation. The strains used were isogenic to the wild-type strain (YMP10381), rad9Δ::LEU2 (YMP10535), rad17Δ::LEU2 (YMP11108), and rad24Δ::TRP1 (YMP10533). HUG1/TUB2 indicates the ratio of the intensity of the HUG1 signal to the TUB2 signal in cells treated with HU or gamma radiation and normalized to the HUG1/TUB2 ratio in control lanes without treatment (lanes 5 and 9 normalized to lane 1, lanes 6 and 10 to lane 2, lanes 7 and 11 to lane 3, and lanes 8 and 12 to lane 4).

FIG. 4

Genetic interactions between HUG1 and MEC1. (A) Deletion of HUG1 suppresses the lethality of mec1Δ. Strains derived from a mating between the hug1Δ (YMB847) and mec1ΔSML1 (pMEC1) (yPP8) strains were plated on control medium YPD and then replica plated to SC-Ura and SC with 5-FOA. The mec1Δ SML1 strain is inviable without the pMEC1 plasmid (pEF208) (growth on SC-Ura, 5-FOA sensitive). The wild-type, hug1Δ and hug1Δ mec1Δ strains can lose the pMEC1 plasmid (no growth on SC-Ura, 5-FOA resistant). (B) Overexpression of HUG1 (pMB379) increases the sensitivity of mec1 sml1-1 (DLY258) mutants to replication arrest, with no effect in the wild-type strain (DLY62). Strains were grown logarithmically in either the absence or presence of HU (0.1 M) for 3.5 h, and 5 μl of a fivefold serial dilution series was plated on SC-Ura with glucose (Glu) or SC-Ura with raffinose plus galactose (Gal).

FIG. 5

HUG1 and SML1 are transcribed independently, and deletions of either gene suppress mec1Δ lethality. The strains used were the wild type (W1588-4A) and the sml1-1 (YEF630), sml1Δ(U952-3C), and hug1Δ2 (YMB847) mutants. Transcription of SML1 was detected in logarithmically grown cells, whereas that of HUG1 was only detected in cells arrested with HU. The sml1-1 mutation (46) deletes a 290-bp region between two direct repeats of 11 bp; the first repeat is 7 bp downstream of the HUG1 stop codon. The sml1-1 mutation is present in most laboratory mec1 strains (25, 46).

FIG. 6

Deletion of HUG1 suppresses the HU sensitivity of the dun1Δ strain. (A) hug1Δ dun1Δ strains are resistant to HU. Spores from tetrad analysis of a mating between the hug1Δ2 (YMB847) and dun1Δ (U971) strains were plated on YPD medium with or without HU (0.1 M). (B) HUG1 restores HU sensitivity in a dun1Δ hug1Δ strain. The hug1Δ dun1Δ spores from panel A were transformed with pMB363 (CEN HUG1 LEU2) or vector alone (pRS315) and plated on SC-Leu with or without HU (0.2 M).

FIG. 7

HUG1 is a critical component of the checkpoint response. Signals received from the sensors for DNA damage and replication arrest are transduced through the kinases MEC1 and TEL1, leading to phosphorylation and activation of RAD53 and DUN1, causing cell cycle arrest and transcriptional induction, which can be DUN1 independent or dependent (12). SML1 (46) and CRT1 (16) function to negatively regulate the MEC1 effectors RNR1 and RNR1 to 4, respectively. Transcription of HUG1 is induced in response to replication arrest and DNA damage in a checkpoint-dependent manner. Deletion of HUG1 rescues the lethality of mec1Δ and the HU sensitivity of dun1Δ strains; overexpression of HUG1 is lethal in combination with a mec1 mutation in the presence of replication arrest or DNA damage. These observations, along with the delayed induction of HUG1 in response to HU, suggest that HUG1 may function, in part, through the negative regulation of MEC1 effectors, perhaps facilitating recovery from the transcriptional response after DNA damage and replication arrest.