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Orchestration of the S-phase and DNA damage checkpoint pathways by replication forks from early origins.

Caldwell JM, Chen Y, Schollaert KL, Theis JF, Babcock GF, Newlon CS, Sanchez Y - J. Cell Biol. (2008)

Bottom Line: We show that providing additional origins activated in early S phase and establishing a paused fork at a replication fork pause site restores S-phase checkpoint signaling to chk1Delta dun1Delta cells and relieves the reliance on the DNA damage checkpoint pathway.Origin licensing and activation are controlled by the cyclin-Cdk complexes.Thus, oncogene-mediated deregulation of cyclins in the early stages of cancer development could contribute to genomic instability through a deficiency in the forks required to establish the S-phase checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.

ABSTRACT
The S-phase checkpoint activated at replication forks coordinates DNA replication when forks stall because of DNA damage or low deoxyribonucleotide triphosphate pools. We explore the involvement of replication forks in coordinating the S-phase checkpoint using dun1Delta cells that have a defect in the number of stalled forks formed from early origins and are dependent on the DNA damage Chk1p pathway for survival when replication is stalled. We show that providing additional origins activated in early S phase and establishing a paused fork at a replication fork pause site restores S-phase checkpoint signaling to chk1Delta dun1Delta cells and relieves the reliance on the DNA damage checkpoint pathway. Origin licensing and activation are controlled by the cyclin-Cdk complexes. Thus, oncogene-mediated deregulation of cyclins in the early stages of cancer development could contribute to genomic instability through a deficiency in the forks required to establish the S-phase checkpoint.

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The timing of origin activation and of the episomal replication fork pause is critical to restore viability to chk1Δ dun1Δ cells. Targeting the acetylase Gcn5p–Gcn4p complex to the late activating origin ARS1412 restored the S-phase checkpoint in chk1D dun1D cells with constitutive expression of Gcn4p. Cells containing a high copy vector expressing Gcn4p and the indicated episomal origin were grown in SC-ura-leu media and spotted onto YPD medium containing the indicated concentration of HU (in millimoles). (bottom) Five representative chk1Δ dun1Δ transformants containing a high copy vector expressing Gcn4p and p12-Ac show results typical for 43/43 chk1Δ dun1Δ transformants containing the same constructs. p12, episome with late activating origin that failed to suppress chk1Δ dun1Δ; p12-Ac, same episome containing binding sites for the Gcn4p–Gcn5p acetylase complex.
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fig5: The timing of origin activation and of the episomal replication fork pause is critical to restore viability to chk1Δ dun1Δ cells. Targeting the acetylase Gcn5p–Gcn4p complex to the late activating origin ARS1412 restored the S-phase checkpoint in chk1D dun1D cells with constitutive expression of Gcn4p. Cells containing a high copy vector expressing Gcn4p and the indicated episomal origin were grown in SC-ura-leu media and spotted onto YPD medium containing the indicated concentration of HU (in millimoles). (bottom) Five representative chk1Δ dun1Δ transformants containing a high copy vector expressing Gcn4p and p12-Ac show results typical for 43/43 chk1Δ dun1Δ transformants containing the same constructs. p12, episome with late activating origin that failed to suppress chk1Δ dun1Δ; p12-Ac, same episome containing binding sites for the Gcn4p–Gcn5p acetylase complex.

Mentions: Because the amount of flanking sequence around ARS1412 is significantly different in p12 and p12ARS, we used p12 (late) to examine whether changing the timing of activation of ARS1412 via a different mechanism was sufficient to restore checkpoint signaling in chk1Δ dun1Δ cells. Recruitment of the acetylase Gcn5p by Gcn4p to the chromosomal ARS1412 was shown to advance its activation timing (Vogelauer et al., 2002). Thus, we inserted Gcn4p binding sites (total of 500 bp) adjacent to ARS1412 in p12 resulting in p12-Ac. Because expression of Gcn4p is induced by amino acid starvation, we introduced a constitutive GCN4 expression construct into the same cells to keep the Gcn4 levels constant in all backgrounds. We observed that the episome with the late origin flanked by Gcn4p–Gcn5p binding sites restored viability to the chk1Δ dun1Δ cells 100% of the time (Fig. 5). These findings suggest that the critical characteristics of the episome that restored viability on HU were the timing of replication fork formation.


Orchestration of the S-phase and DNA damage checkpoint pathways by replication forks from early origins.

Caldwell JM, Chen Y, Schollaert KL, Theis JF, Babcock GF, Newlon CS, Sanchez Y - J. Cell Biol. (2008)

The timing of origin activation and of the episomal replication fork pause is critical to restore viability to chk1Δ dun1Δ cells. Targeting the acetylase Gcn5p–Gcn4p complex to the late activating origin ARS1412 restored the S-phase checkpoint in chk1D dun1D cells with constitutive expression of Gcn4p. Cells containing a high copy vector expressing Gcn4p and the indicated episomal origin were grown in SC-ura-leu media and spotted onto YPD medium containing the indicated concentration of HU (in millimoles). (bottom) Five representative chk1Δ dun1Δ transformants containing a high copy vector expressing Gcn4p and p12-Ac show results typical for 43/43 chk1Δ dun1Δ transformants containing the same constructs. p12, episome with late activating origin that failed to suppress chk1Δ dun1Δ; p12-Ac, same episome containing binding sites for the Gcn4p–Gcn5p acetylase complex.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2290838&req=5

fig5: The timing of origin activation and of the episomal replication fork pause is critical to restore viability to chk1Δ dun1Δ cells. Targeting the acetylase Gcn5p–Gcn4p complex to the late activating origin ARS1412 restored the S-phase checkpoint in chk1D dun1D cells with constitutive expression of Gcn4p. Cells containing a high copy vector expressing Gcn4p and the indicated episomal origin were grown in SC-ura-leu media and spotted onto YPD medium containing the indicated concentration of HU (in millimoles). (bottom) Five representative chk1Δ dun1Δ transformants containing a high copy vector expressing Gcn4p and p12-Ac show results typical for 43/43 chk1Δ dun1Δ transformants containing the same constructs. p12, episome with late activating origin that failed to suppress chk1Δ dun1Δ; p12-Ac, same episome containing binding sites for the Gcn4p–Gcn5p acetylase complex.
Mentions: Because the amount of flanking sequence around ARS1412 is significantly different in p12 and p12ARS, we used p12 (late) to examine whether changing the timing of activation of ARS1412 via a different mechanism was sufficient to restore checkpoint signaling in chk1Δ dun1Δ cells. Recruitment of the acetylase Gcn5p by Gcn4p to the chromosomal ARS1412 was shown to advance its activation timing (Vogelauer et al., 2002). Thus, we inserted Gcn4p binding sites (total of 500 bp) adjacent to ARS1412 in p12 resulting in p12-Ac. Because expression of Gcn4p is induced by amino acid starvation, we introduced a constitutive GCN4 expression construct into the same cells to keep the Gcn4 levels constant in all backgrounds. We observed that the episome with the late origin flanked by Gcn4p–Gcn5p binding sites restored viability to the chk1Δ dun1Δ cells 100% of the time (Fig. 5). These findings suggest that the critical characteristics of the episome that restored viability on HU were the timing of replication fork formation.

Bottom Line: We show that providing additional origins activated in early S phase and establishing a paused fork at a replication fork pause site restores S-phase checkpoint signaling to chk1Delta dun1Delta cells and relieves the reliance on the DNA damage checkpoint pathway.Origin licensing and activation are controlled by the cyclin-Cdk complexes.Thus, oncogene-mediated deregulation of cyclins in the early stages of cancer development could contribute to genomic instability through a deficiency in the forks required to establish the S-phase checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.

ABSTRACT
The S-phase checkpoint activated at replication forks coordinates DNA replication when forks stall because of DNA damage or low deoxyribonucleotide triphosphate pools. We explore the involvement of replication forks in coordinating the S-phase checkpoint using dun1Delta cells that have a defect in the number of stalled forks formed from early origins and are dependent on the DNA damage Chk1p pathway for survival when replication is stalled. We show that providing additional origins activated in early S phase and establishing a paused fork at a replication fork pause site restores S-phase checkpoint signaling to chk1Delta dun1Delta cells and relieves the reliance on the DNA damage checkpoint pathway. Origin licensing and activation are controlled by the cyclin-Cdk complexes. Thus, oncogene-mediated deregulation of cyclins in the early stages of cancer development could contribute to genomic instability through a deficiency in the forks required to establish the S-phase checkpoint.

Show MeSH
Related in: MedlinePlus