scholarly journals N-terminal BRCT domains of the DNA damage checkpoint proteins TOPBP1/Rad4 display distinct specificities for phosphopeptide ligands

2018 ◽  
Author(s):  
Matthew Day ◽  
Mathieu Rappas ◽  
Katie Ptasińska ◽  
Dominik Boos ◽  
Antony W. Oliver ◽  
...  
Keyword(s):  
Dna Damage ◽  
Fission Yeast ◽  
Domain Specificity ◽  
Multiple Partner ◽  
Post Translational Modifications ◽  
Binding Motifs ◽  
Checkpoint Proteins ◽  
High Affinity ◽  
Partner Proteins ◽  
Human Rad9

AbstractTOPBP1 and its fission yeast homologue Rad4, are critical players in a range of DNA replication, repair and damage signalling processes. They are composed of multiple BRCT domains, some of which have the capacity to bind phosphorylated motifs in other proteins. They thus act as multi-point adaptors bringing proteins together into functional combinations, dependent on post-translational modifications downstream of cell cycle and DNA damage signals. We have now structurally and/or biochemically characterised a sufficient number of high-affinity complexes for the conserved N-terminal region of TOPBP1 and Rad4 in complex with diverse phospho-ligands – which include human RAD9 and Treslin, as well as S.pombe Crb2 and Sld3 – to define the key determinants of BRCT domain specificity. We use this information to identify and characterise previously unknown phosphorylation-dependent TOPBP1/Rad4-binding motifs in human RHNO1 and the fission yeast homologue of MDC1, Mdb1. These results provide important insights into how multiple BRCT domains within TOPBP1/Rad4 achieve selective and combinatorial binding of their multiple partner proteins.

scholarly journals BRCT domains of the DNA damage checkpoint proteins TOPBP1/Rad4 display distinct specificities for phosphopeptide ligands

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Matthew Day ◽  
Mathieu Rappas ◽  
Katie Ptasinska ◽  
Dominik Boos ◽  
Antony W Oliver ◽  
...  
Keyword(s):  
Dna Damage ◽  
Fission Yeast ◽  
Domain Specificity ◽  
Editorial Note ◽  
Multiple Partner ◽  
Post Translational Modifications ◽  
Binding Motifs ◽  
Checkpoint Proteins ◽  
Partner Proteins ◽  
Human Rad9

TOPBP1 and its fission yeast homologue Rad4, are critical players in a range of DNA replication, repair and damage signalling processes. They are composed of multiple BRCT domains, some of which bind phosphorylated motifs in other proteins. They thus act as multi-point adaptors bringing proteins together into functional combinations, dependent on post-translational modifications downstream of cell cycle and DNA damage signals. We have now structurally and/or biochemically characterised a sufficient number of high-affinity complexes for the conserved N-terminal region of TOPBP1 and Rad4 with diverse phospho-ligands, including human RAD9 and Treslin, and Schizosaccharomyces pombe Crb2 and Sld3, to define the determinants of BRCT domain specificity. We use this to identify and characterise previously unknown phosphorylation-dependent TOPBP1/Rad4-binding motifs in human RHNO1 and the fission yeast homologue of MDC1, Mdb1. These results provide important insights into how multiple BRCT domains within TOPBP1/Rad4 achieve selective and combinatorial binding of their multiple partner proteins.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see <xref ref-type="decision-letter" rid="SA1">decision letter</xref>).

DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts

2000 ◽  
Vol 349 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Hiroshi MURAKAMI ◽  
Paul NURSE
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Fission Yeast ◽  
Molecular Mechanisms ◽  
Cell Cycle Checkpoint ◽  
Cell Cycle Regulators ◽  
Meiotic Cell ◽  
Checkpoint Proteins ◽  
Dna Damage Checkpoints

The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast.

scholarly journals Physical and functional interactions between MutY glycosylase homologue (MYH) and checkpoint proteins Rad9–Rad1–Hus1

2006 ◽  
Vol 400 (1) ◽  
pp. 53-62 ◽  
Author(s):  
Guoli Shi ◽  
Dau-Yin Chang ◽  
Chih-Chien Cheng ◽  
Xin Guan ◽  
Česlovas Venclovas ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ionizing Radiation ◽  
Binding Site ◽  
Fission Yeast ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Direct Role ◽  
Checkpoint Proteins ◽  
Functional Interactions ◽  
Base Excision

The MYH (MutY glycosylase homologue) increases replication fidelity by removing adenines or 2-hydroxyadenine misincorporated opposite GO (7,8-dihydro-8-oxo-guanine). The 9-1-1 complex (Rad9, Rad1 and Hus1 heterotrimer complex) has been suggested as a DNA damage sensor. Here, we report that hMYH (human MYH) interacts with hHus1 (human Hus1) and hRad1 (human Rad1), but not with hRad9. In addition, interactions between MYH and the 9-1-1 complex, from both the fission yeast Schizosaccharomyces pombe and human cells, are partially interchangeable. The major Hus1-binding site is localized to residues 295–350 of hMYH and to residues 245–293 of SpMYH (S. pombe MYH). Val315 of hMYH and Ile261 of SpMYH play important roles for their interactions with Hus1. hHus1 protein and the 9-1-1 complex of S. pombe can enhance the glycosylase activity of SpMYH. Moreover, the interaction of hMYH–hHus1 is enhanced following ionizing radiation. A significant fraction of the hMYH nuclear foci co-localizes with hRad9 foci in H2O2-treated cells. These results reveal that the 9-1-1 complex plays a direct role in base excision repair.

scholarly journals Fission Yeast Rad17 Associates with Chromatin in Response to Aberrant Genomic Structures

2001 ◽  
Vol 21 (10) ◽  
pp. 3289-3301 ◽  
Author(s):  
Mihoko Kai ◽  
Hiroyuki Tanaka ◽  
Teresa S.-F. Wang
Keyword(s):  
Dna Damage ◽  
Fission Yeast ◽  
Genomic Structure ◽  
Small Subunit ◽  
S Phase ◽  
Dna Integrity ◽  
Checkpoint Proteins ◽  
Defective Mutant ◽  
Mutant Cells

ABSTRACT Fission yeast checkpoint protein Rad17 is required for the DNA integrity checkpoint responses. A fraction of Rad17 is chromatin bound independent of the other checkpoint proteins throughout the cell cycle. Here we show that in response to DNA damage induced by either methyl methanesulfonate treatment or ionizing radiation, increased levels of Rad17 bind to chromatin. Following S-phase stall induced by hydroxyurea or a cdc22 mutation, the chromatin-bound Rad17 progressively dissociates from the chromatin. After S-phase arrest by hydroxyurea in cds1Δ or rad3Δ cells or by replication mutants, Rad17 remains chromatin bound. Rad17 is able to complex in vivo with an Rfc small subunit, Rfc2, but not with Rfc1. Furthermore, cells with rfc1Δ are checkpoint proficient, suggesting that Rfc1 does not have a role in checkpoint function. A checkpoint-defective mutant protein, Rad17(K118E), which has similar nuclear localization to that of the wild type, is unable to bind ATP and has reduced ability in chromatin binding. Mutant Rad17(K118E) protein also has reduced ability to complex with Rfc2, suggesting that Lys118 of Rad17 plays a role in Rad17-Rfc small-subunit complex formation and chromatin association. However, in therad17.K118E mutant cells, Cds1 can be activated by hydroxyurea. Together, these results suggest that Rad17 binds to chromatin in response to an aberrant genomic structure generated from DNA damage, replication mutant arrest, or hydroxyurea arrest in the absence of Cds1. Rad17 is not required to bind chromatin when genomic structures are protected by hydroxyurea-activated Cds1. The possible checkpoint events induced by chromatin-bound Rad17 are discussed.

scholarly journals Crosstalk between the mTOR and DNA Damage Response Pathways in Fission Yeast

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 305
Author(s):  
John-Patrick Alao ◽  
Luc Legon ◽  
Charalampos Rallis
Keyword(s):  
Dna Damage ◽  
Fission Yeast ◽  
Dna Damage Response ◽  
Cell Biology ◽  
Environmental Changes ◽  
Energy Levels ◽  
Protein Translation ◽  
Mtor Pathway ◽  
Age Related ◽  
Damage Response

Cells have developed response systems to constantly monitor environmental changes and accordingly adjust growth, differentiation, and cellular stress programs. The evolutionarily conserved, nutrient-responsive, mechanistic target of rapamycin signaling (mTOR) pathway coordinates basic anabolic and catabolic cellular processes such as gene transcription, protein translation, autophagy, and metabolism, and is directly implicated in cellular and organismal aging as well as age-related diseases. mTOR mediates these processes in response to a broad range of inputs such as oxygen, amino acids, hormones, and energy levels, as well as stresses, including DNA damage. Here, we briefly summarize data relating to the interplays of the mTOR pathway with DNA damage response pathways in fission yeast, a favorite model in cell biology, and how these interactions shape cell decisions, growth, and cell-cycle progression. We, especially, comment on the roles of caffeine-mediated DNA-damage override. Understanding the biology of nutrient response, DNA damage and related pharmacological treatments can lead to the design of interventions towards improved cellular and organismal fitness, health, and survival.

scholarly journals Computational Design of Macrocyclic Binders of S100B(ββ): Novel Peptide Theranostics

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 721
Author(s):  
Srinivasaraghavan Kannan ◽  
Pietro G. A. Aronica ◽  
Thanh Binh Nguyen ◽  
Jianguo Li ◽  
Chandra S. Verma
Keyword(s):  
Molecular Dynamics ◽  
Computational Design ◽  
Diagnostic Tools ◽  
Stapled Peptides ◽  
Altered Expression ◽  
Cellular Processes ◽  
High Affinity ◽  
Limited Success ◽  
Dynamics Simulations ◽  
Partner Proteins

S100B(ββ) proteins are a family of multifunctional proteins that are present in several tissues and regulate a wide variety of cellular processes. Their altered expression levels have been associated with several human diseases, such as cancer, inflammatory disorders and neurodegenerative conditions, and hence are of interest as a therapeutic target and a biomarker. Small molecule inhibitors of S100B(ββ) have achieved limited success. Guided by the wealth of available experimental structures of S100B(ββ) in complex with diverse peptides from various protein interacting partners, we combine comparative structural analysis and molecular dynamics simulations to design a series of peptides and their analogues (stapled) as S100B(ββ) binders. The stapled peptides were subject to in silico mutagenesis experiments, resulting in optimized analogues that are predicted to bind to S100B(ββ) with high affinity, and were also modified with imaging agents to serve as diagnostic tools. These stapled peptides can serve as theranostics, which can be used to not only diagnose the levels of S100B(ββ) but also to disrupt the interactions of S100B(ββ) with partner proteins which drive disease progression, thus serving as novel therapeutics.

scholarly journals Fission Yeast Rad26 Is a Regulatory Subunit of the Rad3 Checkpoint Kinase

2002 ◽  
Vol 13 (2) ◽  
pp. 480-492 ◽  
Author(s):  
Tom D. Wolkow ◽  
Tamar Enoch
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Complex Analysis ◽  
Kinase Activity ◽  
Regulatory Subunit ◽  
Kinase Activation ◽  
Checkpoint Proteins ◽  
Cycle Arrest ◽  
Phosphoinositide 3 Kinase

Fission yeast Rad3 is a member of a family of phosphoinositide 3-kinase -related kinases required for the maintenance of genomic stability in all eukaryotic cells. In fission yeast, Rad3 regulates the cell cycle arrest and recovery activities associated with the G2/M checkpoint. We have developed an assay that directly measures Rad3 kinase activity in cells expressing physiological levels of the protein. Using the assay, we demonstrate directly that Rad3 kinase activity is stimulated by checkpoint signals. Of the five other G2/M checkpoint proteins (Hus1, Rad1, Rad9, Rad17, and Rad26), only Rad26 was required for Rad3 kinase activity. Because Rad26 has previously been shown to interact constitutively with Rad3, our results demonstrate that Rad26 is a regulatory subunit, and Rad3 is the catalytic subunit, of the Rad3/Rad26 kinase complex. Analysis of Rad26/Rad3 kinase activation in rad26.T12, a mutant that is proficient for cell cycle arrest, but defective in recovery, suggests that these two responses to checkpoint signals require quantitatively different levels of kinase activity from the Rad3/Rad26 complex.

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