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The MluI cell cycle box (MCB) motifs, but not damage-responsive elements (DREs), are responsible for the transcriptional induction of the rhp51+ gene in response to DNA replication stress.

Sartagul W, Zhou X, Yamada Y, Ma N, Tanaka K, Furuyashiki T, Ma Y - PLoS ONE (2014)

Bottom Line: Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways.Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+.Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology, Kobe University Graduate School of Medicine, Kobe, Japan.

ABSTRACT
DNA replication stress induces the transcriptional activation of rhp51+, a fission yeast recA homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates rhp51+ transcription is not understood. The promoter region of rhp51+ contains two damage-responsive elements (DREs) and two MluI cell cycle box (MCB) motifs. Using luciferase reporter assays, we examined the role of these elements in rhp51+ transcription. The full-length rhp51+ promoter and a promoter fragment containing MCB motifs only, but not a fragment containing DREs, mediated transcriptional activation upon DNA replication stress. Removal of the MCB motifs from the rhp51+ promoter abolished the induction of rhp51+ transcription by DNA replication stress. Consistent with a role for MCB motifs in rhp51+ transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded rhp51+ transcriptional induction in response to DNA replication stress. Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways. Because MBF is critical for G1/S transcription, we examined how the cell cycle affected rhp51+ transcription. The transcription of rhp51+ and cdc18+, an MBF-dependent G1/S gene, peaked simultaneously in synchronized cdc25-22 cells. Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+. Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.

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Real-time monitoring of rhp51+ gene transcription in wild-type cells treated with HU.A. A schematic diagram of luciferase reporter vectors containing the full-length rhp51+ promoter or rhp51+ promoter deletion mutants. Two DRE decamers are located between bp −234 to −225 (DRE1) and bp −213 to −204 (DRE2) relative to the translation initiation site of the rhp51+ promoter. Two MCB motifs are located between bp −192 to −187 (MCB1) and bp −183 to −178 (MCB2) in the rhp51+ promoter. The following regions of the rhp51+ promoter were inserted upstream of the open reading frame of luciferase: the full-length promoter ranging from bp −345 to −14 (pKB8310, designated Rhp51), a fragment from bp −201 to −14 containing two MCB motifs (pKB8608, designated Rhp51MCB), a fragment from bp −345 to −202 containing two DREs (pKB8606, designated Rhp51DRE), and the full-length promoter from which the two MCB motifs at bp −192 to −178 were deleted (pKB8929, designated Rhp51ΔMCB). B. Effect of HU on promoter activation. Wild-type cells transformed with the full-length rhp51+ (Rhp51), Rhp51MCB, Rhp51DRE, or Rhp51ΔMCB reporter were incubated with luciferin and then treated with HU (1 mM to 4 mM) for real-time monitoring of luciferase activity. Relative light units (RLU) were normalized to the values from wild-type cells harboring the full-length rhp51+ reporter plasmid at 300 min without HU treatment. Representative traces of real-time monitoring are shown in the upper graphs. The lower graphs show the normalized RLU averaged across independent samples at 300 min in cells harboring the indicated reporter plasmids. n = 4 for each group. *P<0.05 and ***P<0.001 compared with the vehicle condition for the respective reporter using one-way ANOVA followed by Tukey's test.
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pone-0111936-g002: Real-time monitoring of rhp51+ gene transcription in wild-type cells treated with HU.A. A schematic diagram of luciferase reporter vectors containing the full-length rhp51+ promoter or rhp51+ promoter deletion mutants. Two DRE decamers are located between bp −234 to −225 (DRE1) and bp −213 to −204 (DRE2) relative to the translation initiation site of the rhp51+ promoter. Two MCB motifs are located between bp −192 to −187 (MCB1) and bp −183 to −178 (MCB2) in the rhp51+ promoter. The following regions of the rhp51+ promoter were inserted upstream of the open reading frame of luciferase: the full-length promoter ranging from bp −345 to −14 (pKB8310, designated Rhp51), a fragment from bp −201 to −14 containing two MCB motifs (pKB8608, designated Rhp51MCB), a fragment from bp −345 to −202 containing two DREs (pKB8606, designated Rhp51DRE), and the full-length promoter from which the two MCB motifs at bp −192 to −178 were deleted (pKB8929, designated Rhp51ΔMCB). B. Effect of HU on promoter activation. Wild-type cells transformed with the full-length rhp51+ (Rhp51), Rhp51MCB, Rhp51DRE, or Rhp51ΔMCB reporter were incubated with luciferin and then treated with HU (1 mM to 4 mM) for real-time monitoring of luciferase activity. Relative light units (RLU) were normalized to the values from wild-type cells harboring the full-length rhp51+ reporter plasmid at 300 min without HU treatment. Representative traces of real-time monitoring are shown in the upper graphs. The lower graphs show the normalized RLU averaged across independent samples at 300 min in cells harboring the indicated reporter plasmids. n = 4 for each group. *P<0.05 and ***P<0.001 compared with the vehicle condition for the respective reporter using one-way ANOVA followed by Tukey's test.

Mentions: A 332-bp DNA fragment in the 5′ flanking region of the rhp51+ gene was amplified using the following PCR primers: sense primer 2737, 5′-AAA ACT GCA GGA CCA GTG CTG TTC TCT TGT TG -3′ and antisense primer 2738, 5′-CCG CTC GAG GCA CGA AAT TAT CAC TAT TCT GG-3′. The amplified products containing the 332-bp rhp51+ promoter (−345 to −14, Figure 1A) were subcloned into a pGL3(R2.2)-basic multicopy vector (Promega) which contains a destabilized luciferase reporter gene, as described previously [17]. The resulting plasmid was registered as pKB8310 and used as the full-length rhp51+ reporter vector. The truncated rhp51+ promoter vectors were constructed as described above except that the 332-bp DNA fragment was replaced by a 145-bp DNA fragment (−345 to −202, containing DRE motifs, Figure 2A) or a 187-bp DNA fragment (−201 to −14, containing MCB motifs, Figure 2A). The resulting plasmids were registered as pKB8606 (Rhp51DRE reporter vector) and pKB8608 (Rhp51MCB reporter vector), respectively. The reporter vector containing three tandem repeats of the MCB motif was constructed as described previously [17], except that the following MCB oligonucleotides were used: sense primer, 5′-GGC TTC GGA CGC GTT ATA CAC GGA CGC GTT ATA CAC ACG GAC GCG TTA GCA C-3′ and antisense primer, 5′- TCG AGT GCA TAA CGC GTC CGT GTG TAT AAC GCG TCC GTG TAT AAC GCG TCC GAA GCC TGC A-3′. The resulting plasmid was registered as pKB8888 (3xMCB reporter vector). The Rhp51ΔMCB reporter vector was constructed using pKB8310 as a template, primer 4730 (5′-CTA GGT AAC AAT TGA TTG AAA TTT AAT TCC TTC ACA ATC CC-3′) as the sense primer, and primer 4731 (5′-GGG ATT GTG AAG GAA TTA AAT TTC AAT CAA TCA ATT GTT ACC TAG-3′) as the antisense primer. The resulting plasmid was registered as pKB8929 (Rhp51ΔMCB reporter vector).


The MluI cell cycle box (MCB) motifs, but not damage-responsive elements (DREs), are responsible for the transcriptional induction of the rhp51+ gene in response to DNA replication stress.

Sartagul W, Zhou X, Yamada Y, Ma N, Tanaka K, Furuyashiki T, Ma Y - PLoS ONE (2014)

Real-time monitoring of rhp51+ gene transcription in wild-type cells treated with HU.A. A schematic diagram of luciferase reporter vectors containing the full-length rhp51+ promoter or rhp51+ promoter deletion mutants. Two DRE decamers are located between bp −234 to −225 (DRE1) and bp −213 to −204 (DRE2) relative to the translation initiation site of the rhp51+ promoter. Two MCB motifs are located between bp −192 to −187 (MCB1) and bp −183 to −178 (MCB2) in the rhp51+ promoter. The following regions of the rhp51+ promoter were inserted upstream of the open reading frame of luciferase: the full-length promoter ranging from bp −345 to −14 (pKB8310, designated Rhp51), a fragment from bp −201 to −14 containing two MCB motifs (pKB8608, designated Rhp51MCB), a fragment from bp −345 to −202 containing two DREs (pKB8606, designated Rhp51DRE), and the full-length promoter from which the two MCB motifs at bp −192 to −178 were deleted (pKB8929, designated Rhp51ΔMCB). B. Effect of HU on promoter activation. Wild-type cells transformed with the full-length rhp51+ (Rhp51), Rhp51MCB, Rhp51DRE, or Rhp51ΔMCB reporter were incubated with luciferin and then treated with HU (1 mM to 4 mM) for real-time monitoring of luciferase activity. Relative light units (RLU) were normalized to the values from wild-type cells harboring the full-length rhp51+ reporter plasmid at 300 min without HU treatment. Representative traces of real-time monitoring are shown in the upper graphs. The lower graphs show the normalized RLU averaged across independent samples at 300 min in cells harboring the indicated reporter plasmids. n = 4 for each group. *P<0.05 and ***P<0.001 compared with the vehicle condition for the respective reporter using one-way ANOVA followed by Tukey's test.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4221157&req=5

pone-0111936-g002: Real-time monitoring of rhp51+ gene transcription in wild-type cells treated with HU.A. A schematic diagram of luciferase reporter vectors containing the full-length rhp51+ promoter or rhp51+ promoter deletion mutants. Two DRE decamers are located between bp −234 to −225 (DRE1) and bp −213 to −204 (DRE2) relative to the translation initiation site of the rhp51+ promoter. Two MCB motifs are located between bp −192 to −187 (MCB1) and bp −183 to −178 (MCB2) in the rhp51+ promoter. The following regions of the rhp51+ promoter were inserted upstream of the open reading frame of luciferase: the full-length promoter ranging from bp −345 to −14 (pKB8310, designated Rhp51), a fragment from bp −201 to −14 containing two MCB motifs (pKB8608, designated Rhp51MCB), a fragment from bp −345 to −202 containing two DREs (pKB8606, designated Rhp51DRE), and the full-length promoter from which the two MCB motifs at bp −192 to −178 were deleted (pKB8929, designated Rhp51ΔMCB). B. Effect of HU on promoter activation. Wild-type cells transformed with the full-length rhp51+ (Rhp51), Rhp51MCB, Rhp51DRE, or Rhp51ΔMCB reporter were incubated with luciferin and then treated with HU (1 mM to 4 mM) for real-time monitoring of luciferase activity. Relative light units (RLU) were normalized to the values from wild-type cells harboring the full-length rhp51+ reporter plasmid at 300 min without HU treatment. Representative traces of real-time monitoring are shown in the upper graphs. The lower graphs show the normalized RLU averaged across independent samples at 300 min in cells harboring the indicated reporter plasmids. n = 4 for each group. *P<0.05 and ***P<0.001 compared with the vehicle condition for the respective reporter using one-way ANOVA followed by Tukey's test.
Mentions: A 332-bp DNA fragment in the 5′ flanking region of the rhp51+ gene was amplified using the following PCR primers: sense primer 2737, 5′-AAA ACT GCA GGA CCA GTG CTG TTC TCT TGT TG -3′ and antisense primer 2738, 5′-CCG CTC GAG GCA CGA AAT TAT CAC TAT TCT GG-3′. The amplified products containing the 332-bp rhp51+ promoter (−345 to −14, Figure 1A) were subcloned into a pGL3(R2.2)-basic multicopy vector (Promega) which contains a destabilized luciferase reporter gene, as described previously [17]. The resulting plasmid was registered as pKB8310 and used as the full-length rhp51+ reporter vector. The truncated rhp51+ promoter vectors were constructed as described above except that the 332-bp DNA fragment was replaced by a 145-bp DNA fragment (−345 to −202, containing DRE motifs, Figure 2A) or a 187-bp DNA fragment (−201 to −14, containing MCB motifs, Figure 2A). The resulting plasmids were registered as pKB8606 (Rhp51DRE reporter vector) and pKB8608 (Rhp51MCB reporter vector), respectively. The reporter vector containing three tandem repeats of the MCB motif was constructed as described previously [17], except that the following MCB oligonucleotides were used: sense primer, 5′-GGC TTC GGA CGC GTT ATA CAC GGA CGC GTT ATA CAC ACG GAC GCG TTA GCA C-3′ and antisense primer, 5′- TCG AGT GCA TAA CGC GTC CGT GTG TAT AAC GCG TCC GTG TAT AAC GCG TCC GAA GCC TGC A-3′. The resulting plasmid was registered as pKB8888 (3xMCB reporter vector). The Rhp51ΔMCB reporter vector was constructed using pKB8310 as a template, primer 4730 (5′-CTA GGT AAC AAT TGA TTG AAA TTT AAT TCC TTC ACA ATC CC-3′) as the sense primer, and primer 4731 (5′-GGG ATT GTG AAG GAA TTA AAT TTC AAT CAA TCA ATT GTT ACC TAG-3′) as the antisense primer. The resulting plasmid was registered as pKB8929 (Rhp51ΔMCB reporter vector).

Bottom Line: Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways.Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+.Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology, Kobe University Graduate School of Medicine, Kobe, Japan.

ABSTRACT
DNA replication stress induces the transcriptional activation of rhp51+, a fission yeast recA homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates rhp51+ transcription is not understood. The promoter region of rhp51+ contains two damage-responsive elements (DREs) and two MluI cell cycle box (MCB) motifs. Using luciferase reporter assays, we examined the role of these elements in rhp51+ transcription. The full-length rhp51+ promoter and a promoter fragment containing MCB motifs only, but not a fragment containing DREs, mediated transcriptional activation upon DNA replication stress. Removal of the MCB motifs from the rhp51+ promoter abolished the induction of rhp51+ transcription by DNA replication stress. Consistent with a role for MCB motifs in rhp51+ transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded rhp51+ transcriptional induction in response to DNA replication stress. Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways. Because MBF is critical for G1/S transcription, we examined how the cell cycle affected rhp51+ transcription. The transcription of rhp51+ and cdc18+, an MBF-dependent G1/S gene, peaked simultaneously in synchronized cdc25-22 cells. Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+. Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.

Show MeSH
Related in: MedlinePlus