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The role of specific checkpoint-induced S-phase transcripts in resistance to replicative stress.

Dutta C, Rhind N - PLoS ONE (2009)

Bottom Line: In response to replication arrest, the fission yeast Cds1 checkpoint kinase maintains the normal S-phase transcriptional program by regulating MBF, the S-phase transcription factor.Finally, we investigate the roles of two specific transcripts: mik1 and mrc1.These results demonstrate the general importance of checkpoint regulation of G1/S transcription in response to replicative stress and elucidate the specific roles of Mik1 and Mrc1 in the checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Checkpoint activation during S phase modulates transcription. In response to replication arrest, the fission yeast Cds1 checkpoint kinase maintains the normal S-phase transcriptional program by regulating MBF, the S-phase transcription factor. We show that similar regulation occurs in response to DNA damage during S-phase. We test the relative contributions to replication-stress resistance of transcriptional regulation and the two other major checkpoint functions: cell-cycle arrest and fork stabilization. We show that, although transcriptional regulation provides only modest resistance relative to fork stabilization, it contributes significantly to cell survival. Finally, we investigate the roles of two specific transcripts: mik1 and mrc1. These results demonstrate the general importance of checkpoint regulation of G1/S transcription in response to replicative stress and elucidate the specific roles of Mik1 and Mrc1 in the checkpoint.

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mrc1 plays major role in recovery from replicative stress in rad3Δ cdc10-2E cells.rad3Δ (yFS628), rad3Δ cdc10-2E (yFS630), rad3Δ cdc10-2E mrc1Δ (yFS645) and rad3Δ </emph>cdc10-2E mik1</emph>Δ (yFS643) cells were treated with 10 mM HU for 4 hours, washed, resuspended in fresh media and collected every 20 minutes for flow cytometry. S-phase progression was calculated by measuring the mean of the S-phase peak as a percentage of the position of between the means of the 1C and 2C contents.
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pone-0006944-g006: mrc1 plays major role in recovery from replicative stress in rad3Δ cdc10-2E cells.rad3Δ (yFS628), rad3Δ cdc10-2E (yFS630), rad3Δ cdc10-2E mrc1Δ (yFS645) and rad3Δ </emph>cdc10-2E mik1</emph>Δ (yFS643) cells were treated with 10 mM HU for 4 hours, washed, resuspended in fresh media and collected every 20 minutes for flow cytometry. S-phase progression was calculated by measuring the mean of the S-phase peak as a percentage of the position of between the means of the 1C and 2C contents.

Mentions: Mrc1 has two functions: it plays an important role in transmitting checkpoint signaling during S phase and it has an important checkpoint-independent role during replication [14], [17]. To test the hypothesis that constitutive expression of mrc1 contributes to a checkpoint-independent stabilization of replication forks, we assayed for recovery of replication from an HU arrest (Figure 6). We treated asynchronous cultures with 10 mM HU for 4 hours, released them in fresh media and collected sample for flow cytometry for every 20 minutes. The wild type cells and cdc10-2E cells finished replication within 60 minutes after release from HU block. rad3Δ cells do not recover well from HU arrest, achieving an average of less than 40% replicated after 180 minutes. rad3Δ cdc10-2E cells recover better from HU arrest, replicating faster than rad3Δ cell and reaching greater than 50% replicated by 180 minutes. To determine the beneficial role of mik1 and mrc1 in rad3Δ cdc10-2E cells, we performed the same experiment with rad3Δ cdc10-2E mrc1Δ and rad3Δ cdc10-2E mik1Δ cells. We observed S-phase progression in rad3Δ cdc10-2E mik1Δ cells similar to rad3Δ cdc10-2E. However S-phase progression in rad3Δ cdc10-2E mrc1Δ cells is similar to rad3Δ alone, suggesting that the increased recovery provided by transcription of MBF target genes is due primarily to the expression of mrc1.


The role of specific checkpoint-induced S-phase transcripts in resistance to replicative stress.

Dutta C, Rhind N - PLoS ONE (2009)

mrc1 plays major role in recovery from replicative stress in rad3Δ cdc10-2E cells.rad3Δ (yFS628), rad3Δ cdc10-2E (yFS630), rad3Δ cdc10-2E mrc1Δ (yFS645) and rad3Δ </emph>cdc10-2E mik1</emph>Δ (yFS643) cells were treated with 10 mM HU for 4 hours, washed, resuspended in fresh media and collected every 20 minutes for flow cytometry. S-phase progression was calculated by measuring the mean of the S-phase peak as a percentage of the position of between the means of the 1C and 2C contents.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2735676&req=5

pone-0006944-g006: mrc1 plays major role in recovery from replicative stress in rad3Δ cdc10-2E cells.rad3Δ (yFS628), rad3Δ cdc10-2E (yFS630), rad3Δ cdc10-2E mrc1Δ (yFS645) and rad3Δ </emph>cdc10-2E mik1</emph>Δ (yFS643) cells were treated with 10 mM HU for 4 hours, washed, resuspended in fresh media and collected every 20 minutes for flow cytometry. S-phase progression was calculated by measuring the mean of the S-phase peak as a percentage of the position of between the means of the 1C and 2C contents.
Mentions: Mrc1 has two functions: it plays an important role in transmitting checkpoint signaling during S phase and it has an important checkpoint-independent role during replication [14], [17]. To test the hypothesis that constitutive expression of mrc1 contributes to a checkpoint-independent stabilization of replication forks, we assayed for recovery of replication from an HU arrest (Figure 6). We treated asynchronous cultures with 10 mM HU for 4 hours, released them in fresh media and collected sample for flow cytometry for every 20 minutes. The wild type cells and cdc10-2E cells finished replication within 60 minutes after release from HU block. rad3Δ cells do not recover well from HU arrest, achieving an average of less than 40% replicated after 180 minutes. rad3Δ cdc10-2E cells recover better from HU arrest, replicating faster than rad3Δ cell and reaching greater than 50% replicated by 180 minutes. To determine the beneficial role of mik1 and mrc1 in rad3Δ cdc10-2E cells, we performed the same experiment with rad3Δ cdc10-2E mrc1Δ and rad3Δ cdc10-2E mik1Δ cells. We observed S-phase progression in rad3Δ cdc10-2E mik1Δ cells similar to rad3Δ cdc10-2E. However S-phase progression in rad3Δ cdc10-2E mrc1Δ cells is similar to rad3Δ alone, suggesting that the increased recovery provided by transcription of MBF target genes is due primarily to the expression of mrc1.

Bottom Line: In response to replication arrest, the fission yeast Cds1 checkpoint kinase maintains the normal S-phase transcriptional program by regulating MBF, the S-phase transcription factor.Finally, we investigate the roles of two specific transcripts: mik1 and mrc1.These results demonstrate the general importance of checkpoint regulation of G1/S transcription in response to replicative stress and elucidate the specific roles of Mik1 and Mrc1 in the checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Checkpoint activation during S phase modulates transcription. In response to replication arrest, the fission yeast Cds1 checkpoint kinase maintains the normal S-phase transcriptional program by regulating MBF, the S-phase transcription factor. We show that similar regulation occurs in response to DNA damage during S-phase. We test the relative contributions to replication-stress resistance of transcriptional regulation and the two other major checkpoint functions: cell-cycle arrest and fork stabilization. We show that, although transcriptional regulation provides only modest resistance relative to fork stabilization, it contributes significantly to cell survival. Finally, we investigate the roles of two specific transcripts: mik1 and mrc1. These results demonstrate the general importance of checkpoint regulation of G1/S transcription in response to replicative stress and elucidate the specific roles of Mik1 and Mrc1 in the checkpoint.

Show MeSH
Related in: MedlinePlus