Dataset Information

Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast.

ABSTRACT: Phosphorylation of histone H2A or H2AX is an early and sensitive marker of DNA damage in eukaryotic cells, although mutation of the conserved damage-dependent phosphorylation site is well tolerated. Here, we show that H2A phosphorylation is required for cell-cycle arrest in response to DNA damage at the G1/S transition in budding yeast. Furthermore, we show that the tandem BRCT domain of Rad9 interacts directly with phosphorylated H2A in vitro and that a rad9 point mutation that abolishes this interaction results in in vivo phenotypes that are similar to those caused by an H2A phosphorylation site mutation. Remarkably, similar checkpoint defects are also caused by a Rad9 Tudor domain mutation that impairs Rad9 chromatin association already in undamaged cells. These findings indicate that constitutive Tudor domain-mediated and damage-specific BRCT domain-phospho-H2A-dependent interactions of Rad9 with chromatin cooperate to establish G1 checkpoint arrest.

SUBMITTER: Hammet A

PROVIDER: S-EPMC1973948 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast.

Hammet Andrew A Magill Christine C Heierhorst Jörg J Jackson Stephen P SP

EMBO reports 20070803 9

Phosphorylation of histone H2A or H2AX is an early and sensitive marker of DNA damage in eukaryotic cells, although mutation of the conserved damage-dependent phosphorylation site is well tolerated. Here, we show that H2A phosphorylation is required for cell-cycle arrest in response to DNA damage at the G1/S transition in budding yeast. Furthermore, we show that the tandem BRCT domain of Rad9 interacts directly with phosphorylated H2A in vitro and that a rad9 point mutation that abolishes this i ...[more]

PMID: 17721446

Similar Datasets

Project description:In Saccharomyces cerevisiae the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Fkh1 binds directly to origin-adjacent Fkh1 binding sites (FKH sites), providing evidence that Fkh1 acts directly. However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are undefined. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module that binds phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex) -origin binding step, the G1-phase event that initiates the origin cycle. The relevance of the Fkh1-FHA to either chromosomal replication or ORC-origin interactions at genome scale has not been reported. Here, S-phase SortSeq experiments were used to assess genome replication in proliferating FKH1 and fkh1R80A mutant cells. The data provided evidence that the Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. ORC ChIPSeq data provided evidence that the Fkh1-FHA domain promoted normal ORC-origin binding at FHA-stimulated origins. Origins are associated with a distinctive nucleosome organization that frames a nucleosome deplected region (NDR) over the origin, with ORC contributing to this architecture. To ask whether the Fkh1-FHA domain had an impact on the ORC and nucleosome protein-DNA interactions at origins, MNaseSeq data were generated from G1-arrested and proliferating FKH1 and fkh1R80A mutant cell populations. These data provided evidence that origin groups differentially regulated by the Fkh1-FHA domain were characterized by distinct G1-phase ORC and nucleosome configurations that were Fkh1FHA-dependent. Thus, the Fkh1-FHA domain performed an important post-DNA binding role of the Fkh1 protein in promoting hallmarks of origin-associated protein-DNA architecture in G1-phase and the activity of early origins in S-phase.

Project description:In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.

Dataset Information

Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast.

Publications

Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets