Novel Role for Checkpoint Rad53 Protein Kinase in the Initiation of Chromosomal DNA Replication in<i>Saccharomyces cerevisiae</i>

Dohrmann, Paul R.; Sclafani, Robert A.

doi:10.1534/genetics.106.060236

Cited by 26 publications

(57 citation statements)

References 81 publications

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“…Although the Dbf4-ND109 protein both binds to and activates Cdc7 normally , it is defective for binding Cdc5 and Rad53 (this study). Therefore, we tested whether the synthetic lethality between dbf4-NΔ109 and rad53-1 was due to the loss of the Dbf4-Cdc5 or Dbf4-Rad53 interactions.We first sequenced the rad53-1 gene (Weinert et al 1994) and found a single G653E point mutation, which is identical to that reported for the rad53-11 allele (Dohrmann and Sclafani 2006). G653 falls within a loop between the b6 and b7 strands of the FHA2 domain ( Figure 5A) and is adjacent to the conserved N655 residue, which plays an important role in substrate recognition (Byeon et al 2001).…”

supporting

confidence: 66%

“…We first sequenced the rad53-1 gene (Weinert et al 1994) and found a single G653E point mutation, which is identical to that reported for the rad53-11 allele (Dohrmann and Sclafani 2006). G653 falls within a loop between the b6 and b7 strands of the FHA2 domain ( Figure 5A) and is adjacent to the conserved N655 residue, which plays an important role in substrate recognition (Byeon et al 2001).…”

supporting

confidence: 66%

“…Rad53 was present in the second IP (Figure 4, right), indicating that Rad53 forms a ternary complex with Cdc5 and DDK. Together, these results demonstrate that the Dbf4 N terminus acts as a docking site for both Rad53 and Cdc5 and that both kinases can simultaneously associate with DDK.Rad53 checkpoint defect together with loss of specific Dbf4 N-terminal residues results in synthetic lethality DDK and rad53 mutants show a series of complex genetic interactions (Desany et al 1998;Dohrmann et al 1999;Dohrmann and Sclafani 2006;. We previously reported that the dbf4-NΔ109 mutant was synthetically lethal with the rad53-1 hypomorphic mutant .…”

mentioning

confidence: 78%

“…Rad53 checkpoint defect together with loss of specific Dbf4 N-terminal residues results in synthetic lethality DDK and rad53 mutants show a series of complex genetic interactions (Desany et al 1998;Dohrmann et al 1999;Dohrmann and Sclafani 2006;. We previously reported that the dbf4-NΔ109 mutant was synthetically lethal with the rad53-1 hypomorphic mutant .…”

Section: Dbf4-fha1 Domain Interaction Is Phospho-threonine Dependentmentioning

confidence: 98%

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DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

et al. 2013

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Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK-and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5-and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.T HE fidelity of chromosome replication depends on checkpoint mechanisms to stabilize stalled forks, regulate origin activation, and repair DNA damage (Hartwell and Weinert 1989;Bartek et al. 2004;Segurado and Tercero 2009). In response to replication stress, the replication checkpoint maintains replisome stability and prevents late origins from firing, which allows time for DNA repair and the completion of DNA replication prior to chromosome segregation. Incomplete DNA replication or uncoordinated origin firing following DNA damage can result in genomic instability, cancer predisposition, and premature aging (Branzei and Foiani 2010).In the budding yeast Saccharomyces cerevisiae, activation of the checkpoint sensor kinase Mec1 (vertebrate ATR, Ataxia Telangiectasia and Rad3-related) is triggered at stalled forks or sites of DNA damage (Majka et al. 2006;Labib and De Piccoli 2011). Subsequent signal amplification through the Mrc1 or Rad9 adaptors leads to activation of the checkpoint kinase Rad53 (the ortholog of the human tumor suppressor Chk2) (Branzei and Foiani 2009). Rad53 is an integral transducer of various cellular responses to replication stress or DNA damage. Rad53 induces a series of transcriptional responses through MBF-regulated genes (Bastos de Oliveira et al. 2012;Travesa et al. 2012) and also activates the Dun1 kinase, which promotes the expression of ribonucleotide reductase (RNR) subunits and additional DNA repair genes (Huang et al. 1998). In parallel, Rad53 down-regulates the RNR inhibitor Sml1 to increase deoxyribonucleotide levels and facilitate DNA synthesis (Zhao et al. 2001). In response to replication fork stalling, Rad53 prevents the activation of late replication origins by phosphorylating two proteins required for the initiation of DNA replication: Dbf4 and Sld3 (...

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supporting

confidence: 66%

supporting

confidence: 66%

mentioning

confidence: 78%

Section: Dbf4-fha1 Domain Interaction Is Phospho-threonine Dependentmentioning

confidence: 98%

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DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

et al. 2013

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“…As an initial approach to understanding how srp1 mutants might affect DNA replication, we utilized a plasmid loss assay (Friedman et al 1996;Dohrmann and Sclafani 2006). This assay monitors loss of plasmids containing an ARS, which function as replication origins in S. cerevisiae (Dohrmann and Sclafani 2006).…”

Section: Resultsmentioning

confidence: 99%

The Classical Nuclear Localization Signal Receptor, Importin-α, Is Required for Efficient Transition Through the G1/S Stage of the Cell Cycle inSaccharomyces cerevisiae

et al. 2009

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There is significant evidence linking nucleocytoplasmic transport to cell cycle control. The budding yeast, Saccharomyces cerevisiae, serves as an ideal model system for studying transport events critical to cell cycle progression because the nuclear envelope remains intact throughout the cell cycle. Previous studies linked the classical nuclear localization signal (cNLS) receptor, importin-a/Srp1, to the G 2 /M transition of the cell cycle. Here, we utilize two engineered mutants of importin-a/Srp1 with specific molecular defects to explore how protein import affects cell cycle progression. One mutant, Srp1-E402Q, is defective in binding to cNLS cargoes that contain two clusters of basic residues termed a bipartite cNLS. The other mutant, Srp1-55, has defects in release of cNLS cargoes into the nucleus. Consistent with distinct in vivo functional consequences for each of the Srp1 mutants analyzed, we find that overexpression of different nuclear transport factors can suppress the temperature-sensitive growth defects of each mutant. Studies aimed at understanding how each of these mutants affects cell cycle progression reveal a profound defect at the G 1 to S phase transition in both srp1-E402Q and srp1-55 mutants as well as a modest G 1 /S defect in the temperature-sensitive srp1-31 mutant, which was previously implicated in G 2 /M. We take advantage of the characterized defects in the srp1-E402Q and srp1-55 mutants to predict candidate cargo proteins likely to be affected in these mutants and provide evidence that three of these cargoes, Cdc45, Yox1, and Mcm10, are not efficiently localized to the nucleus in importin-a mutants. These results reveal that the classical nuclear protein import pathway makes important contributions to the G 1 /S cell cycle transition.

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Novel Role for Checkpoint Rad53 Protein Kinase in the Initiation of Chromosomal DNA Replication inSaccharomyces cerevisiae

Cited by 26 publications

References 81 publications

DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

The Classical Nuclear Localization Signal Receptor, Importin-α, Is Required for Efficient Transition Through the G1/S Stage of the Cell Cycle inSaccharomyces cerevisiae

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