Colocalization of Sensors Is Sufficient to Activate the DNA Damage Checkpoint in the Absence of Damage

Bonilla, Carla Yaneth; Melo, Justine A.; Toczyski, David P.

doi:10.1016/j.molcel.2008.03.023

Cited by 153 publications

(188 citation statements)

References 70 publications

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“…The importance of a specific protein-protein association in DNA damage checkpoint signaling is further revealed by the observations that a forced assembly of signaling proteins to the same location in the genome could activate Rad53 without introducing DNA damage signals (41,42). In addition, many proteins are known to localize to the site of DNA damage in cells (43).…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Chen¹,

Zhou

2009

Journal of Biological Chemistry

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Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint.Faithful replication of the genome is important for the survival of all organisms. During DNA replication, replication stress can arise from a variety of situations, including intrinsic errors made by DNA polymerases, difficulties in replicating repeated DNA sequences, and failures to repair damaged DNA caused by either endogenous oxidative agents or exogenous mutagens such as UV light and DNA-damaging chemicals (1-3). In eukaryotes, there is an evolutionarily conserved DNA replication checkpoint that becomes activated in response to DNA replication stress. It helps to stabilize DNA replication forks, block late replication origin firing, and delay mitosis and ultimately helps recovery from stalled replication forks after DNA repair (4 -7). Defects in the DNA replication checkpoint could result in elevated genomic instabilities, cancer development, or cell death (8, 9).Aside from replicating the genome, the DNA replication forks also provide a platform to assemble many signaling proteins that function in the DNA replication checkpoint. In the budding yeast Saccharomyces cerevisiae, Mec1, an ortholog of human ATR, 2 is a phosphoinositide 3-kinase-like kinase (PIKK) involved in sensing stalled DNA replication forks. Mec1 forms a protein complex with Ddc2 (ortholog of human ATRIP). The Mec1-Ddc2 complex is recruited to stalled replication forks through replication protein A (RPA)-coated single-stranded DNA (10, 11). The Mec3-Rad17-Ddc1 complex, a proliferating cell nuclear antigen (PCNA)-like checkpoint clamp and ortholog of the human 9-1-1 complex, was shown to be loaded onto the single-and double-stranded DNA junction of the stalled replication forks by the clamp loader Rad24-RFC complex (12). Once loaded, the Mec3-Rad17-Ddc1 complex stimulates Mec1 kinase activity (13). Dbp11 and its homolog TopBP1 in vertebrates are known components of the replication machinery (14). In addition to regulating the initiation of DNA replication, they were foun...

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Section: Discussionmentioning

confidence: 99%

Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Chen¹,

Zhou

2009

Journal of Biological Chemistry

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“…The activation of Mec1-Ddc2 (ATR-ATRIP in mammals) is regulated by a number of factors: First, Mec1 signaling is dependent on colocalization of the Mec1-Ddc2 with the PCNA-like 9-1-1 Ddc1-Rad17-Mec3 complex (RAD9-RAD1-HUS1, in mammals) (Parrilla-Castellar et al 2004;Bonilla et al 2008). Second, it has been reported that the yeast 9-1-1 complex can activate Mec1 directly in vitro (Majka et al 2006).…”

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confidence: 99%

Regulation of Mec1 kinase activity by the SWI/SNF chromatin remodeling complex

et al. 2015

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ATP-dependent chromatin remodeling complexes alter chromatin structure through interactions with chromatin substrates such as DNA, histones, and nucleosomes. However, whether chromatin remodeling complexes have the ability to regulate nonchromatin substrates remains unclear. Saccharomyces cerevisiae checkpoint kinase Mec1 (ATR in mammals) is an essential master regulator of genomic integrity. Here we found that the SWI/SNF chromatin remodeling complex is capable of regulating Mec1 kinase activity. In vivo, Mec1 activity is reduced by the deletion of Snf2, the core ATPase subunit of the SWI/SNF complex. SWI/SNF interacts with Mec1, and cross-linking studies revealed that the Snf2 ATPase is the main interaction partner for Mec1. In vitro, SWI/SNF can activate Mec1 kinase activity in the absence of chromatin or known activators such as Dpb11. The subunit requirement of SWI/SNFmediated Mec1 regulation differs from that of SWI/SNF-mediated chromatin remodeling. Functionally, SWI/SNFmediated Mec1 regulation specifically occurs in S phase of the cell cycle. Together, these findings identify a novel regulator of Mec1 kinase activity and suggest that ATP-dependent chromatin remodeling complexes can regulate nonchromatin substrates such as a checkpoint kinase.

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“…The generation of single-stranded DNA coated with the single-stranded binding protein RPA 3 is a critical step in the recruitment of Mec1 to sites of damage, which is mediated through Ddc2-RPA interactions (4,5). One or more Mec1 activators are also recruited to these sites, and the juxtaposition on chromatin of a given activator with Mec1 results in the activation of Mec1 protein kinase activity (6) and the phosphorylation of a large number of proteins, including RPA, subunits of the 9-1-1 checkpoint clamp, mediator proteins, and downstream effector kinases such as Rad53.…”

mentioning

confidence: 99%

The Unstructured C-terminal Tail of Yeast Dpb11 (Human TopBP1) Protein Is Dispensable for DNA Replication and the S Phase Checkpoint but Required for the G2/M Checkpoint

Navadgi-Patil¹,

Kumar²,

Burgers

2011

Journal of Biological Chemistry

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Background: Specific activators turn on Mec1/ATR kinase during DNA damage or replication stress, mediating cell cycle arrest. Results: We have separated the replication function of multifunctional Dpb11 from its checkpoint activation function. Conclusion: Dpb11 functions primarily during the G 2 /M phase. Significance: Our results suggest that each phase of the cell cycle may use specific Mec1 activators.

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Colocalization of Sensors Is Sufficient to Activate the DNA Damage Checkpoint in the Absence of Damage

Cited by 153 publications

References 70 publications

Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Regulation of Mec1 kinase activity by the SWI/SNF chromatin remodeling complex

The Unstructured C-terminal Tail of Yeast Dpb11 (Human TopBP1) Protein Is Dispensable for DNA Replication and the S Phase Checkpoint but Required for the G2/M Checkpoint

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