Cooperative Control of Crb2 by ATM Family and Cdc2 Kinases Is Essential for the DNA Damage Checkpoint in Fission Yeast

ABSTRACT The cellular responses to double-stranded breaks (DSBs) typically involve the extensive accumulation of checkpoint proteins in chromatin surrounding the damaged DNA. One well-characterized example involves the checkpoint protein Crb2 in the fission yeast Schizosaccharomyces pombe. The accumulation of Crb2 at DSBs requires the C-terminal phosphorylation of histone H2A (known as γ-H2A) by ATM family kinases in chromatin surrounding the break. It also requires the constitutive methylation of histone H4 on lysine-20 (K20). Interestingly, neither type of histone modification is essential for the Crb2-dependent checkpoint response. However, H4-K20 methylation is essential in a crb2-T215A strain that lacks a cyclin-dependent kinase phosphorylation site in Crb2. Here we explain this genetic interaction by describing a previously overlooked effect of the crb2-T215A mutation. We show that crb2-T215A cells are able to initiate but not sustain a checkpoint response. We also report that γ-H2A is essential for the DNA damage checkpoint in crb2-T215A cells. Importantly, we show that inactivation of Cdc2 in γ-H2A-defective cells impairs Crb2-dependent signaling to the checkpoint kinase Chk1. These findings demonstrate that full Crb2 activity requires phosphorylation of threonine-215 by Cdc2. This regulation of Crb2 is independent of the histone modifications that are required for the hyperaccumulation of Crb2 at DSBs.

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BRCT Domain Interactions with Phospho-Histone H2A Target Crb2 to Chromatin at Double-Strand Breaks and Maintain the DNA Damage Checkpoint

Molecular and Cellular Biology ◽

10.1128/mcb.00413-10 ◽

2010 ◽

Vol 30 (19) ◽

pp. 4732-4743 ◽

Cited By ~ 20

Author(s):

Sevil Sofueva ◽

Li-Lin Du ◽

Oliver Limbo ◽

Jessica S. Williams ◽

Paul Russell

Keyword(s):

Dna Damage ◽

Phosphorylation Site ◽

Binding Pocket ◽

Double Strand Breaks ◽

Histone H2a ◽

Histone H4 ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Domain Interactions ◽

And Function

ABSTRACT Relocalization of checkpoint proteins to chromatin flanking DNA double-strand breaks (DSBs) is critical for cellular responses to DNA damage. Schizosaccharomyces pombe Crb2, which mediates Chk1 activation by Rad3ATR, forms ionizing radiation-induced nuclear foci (IRIF). Crb2 C-terminal BRCT domains (BRCT2) bind histone H2A phosphorylated at a C-terminal SQ motif by Tel1ATM and Rad3ATR, although the functional significance of this interaction is controversial. Here, we show that polar interactions of Crb2 serine-548 and lysine-619 with the phosphate group of phospho-H2A (γ-H2A) are critical for Crb2 IRIF formation and checkpoint function. Mutations of these BRCT2 domain residues have additive effects when combined in a single allele. Combining either mutation with an allele that eliminates the threonine-215 cyclin-dependent kinase phosphorylation site completely abrogates Crb2 IRIF and function. We propose that cooperative phosphate interactions in the BRCT2 γ-H2A-binding pocket of Crb2, coupled with tudor domain interactions with lysine-20 dimethylation of histone H4, facilitate stable recruitment of Crb2 to chromatin surrounding DSBs, which in turn mediates efficient phosphorylation of Chk1 that is required for a sustained checkpoint response. This mechanism of cooperative interactions with the γ-H2A/X phosphate is likely conserved in S. pombe Brc1 and human Mdc1 genome maintenance proteins.

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Regulation of Septum Formation in Aspergillus nidulans by a DNA Damage Checkpoint Pathway

Genetics ◽

10.1093/genetics/148.3.1055 ◽

1998 ◽

Vol 148 (3) ◽

pp. 1055-1067

Author(s):

Steven D Harris ◽

Peter R Kraus

Keyword(s):

Dna Damage ◽

Aspergillus Nidulans ◽

Cellular Response ◽

Dna Damage Checkpoint ◽

Temperature Sensitive ◽

Dna Metabolism ◽

Chromosomal Dna ◽

Checkpoint Response ◽

Septum Formation ◽

Checkpoint Pathway

Abstract In Aspergillus nidulans, germinating conidia undergo multiple rounds of nuclear division before the formation of the first septum. Previous characterization of temperature-sensitive sepB and sepJ mutations showed that although they block septation, they also cause moderate defects in chromosomal DNA metabolism. Results presented here demonstrate that a variety of other perturbations of chromosomal DNA metabolism also delay septum formation, suggesting that this is a general cellular response to the presence of sublethal DNA damage. Genetic evidence is provided that suggests that high levels of cyclin-dependent kinase (cdk) activity are required for septation in A. nidulans. Consistent with this notion, the inhibition of septum formation triggered by defects in chromosomal DNA metabolism depends upon Tyr-15 phosphorylation of the mitotic cdk p34nimX. Moreover, this response also requires elements of the DNA damage checkpoint pathway. A model is proposed that suggests that the DNA damage checkpoint response represents one of multiple sensory inputs that modulates p34nimX activity to control the timing of septum formation.

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Cell cycle G2 arrest induced by HIV-1 Vpr in fission yeast (Schizosaccharomyces pombe) is independent of cell death and early genes in the DNA damage checkpoint

Virus Research ◽

10.1016/s0168-1702(00)00167-2 ◽

2000 ◽

Vol 68 (2) ◽

pp. 161-173 ◽

Cited By ~ 30

Author(s):

Robert T. Elder ◽

Min Yu ◽

Mingzhong Chen ◽

Steven Edelson ◽

Yuqi Zhao

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Death ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Dna Damage Checkpoint ◽

G2 Arrest ◽

Early Genes ◽

Hiv 1

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Monitoring S. pombe genome stress by visualizing end-binding protein Ku

10.1101/2020.06.12.149054 ◽

2020 ◽

Author(s):

Chance Jones ◽

Susan L Forsburg

Keyword(s):

Dna Damage ◽

Fission Yeast ◽

Protein Complex ◽

Genome Stability ◽

Binding Protein ◽

Dna Lesions ◽

Live Cells ◽

Dna Damage Checkpoint ◽

Unique Pattern ◽

Ku Protein

AbstractStudies of genome stability have exploited visualization of fluorescently tagged proteins in live cells to characterize DNA damage, checkpoint, and repair responses. In this report, we describe a new tool for fission yeast, a tagged version of the end-binding protein Pku70 which is part of the KU protein complex. We compare Pku70 localization to other markers upon treatment to various genotoxins, and identify a unique pattern of distribution. Pku70 provides a new tool to define and characterize DNA lesions and the repair response.

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A DNA damage checkpoint response in telomere-initiated senescence

Nature ◽

10.1038/nature02118 ◽

2003 ◽

Vol 426 (6963) ◽

pp. 194-198 ◽

Cited By ~ 1632

Author(s):

Fabrizio d'Adda di Fagagna ◽

Philip M. Reaper ◽

Lorena Clay-Farrace ◽

Heike Fiegler ◽

Philippa Carr ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Checkpoint ◽

Checkpoint Response

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Abstract 4197: A DNA damage checkpoint response to unrepaired ultraviolet radiation-induced lesions which is defective in melanoma

10.1158/1538-7445.am2011-4197 ◽

2011 ◽

Author(s):

Matthew Wigan ◽

Sandra Pavey ◽

Kelly Brooks ◽

Nichole Giles ◽

Andrew Burgess ◽

...

Keyword(s):

Dna Damage ◽

Ultraviolet Radiation ◽

Dna Damage Checkpoint ◽

Checkpoint Response ◽

Radiation Induced

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DNA Damage Checkpoint Control of Mitosis in Fission Yeast

Cold Spring Harbor Symposia on Quantitative Biology ◽

10.1101/sqb.2000.65.353 ◽

2000 ◽

Vol 65 (0) ◽

pp. 353-360 ◽

Cited By ~ 3

Author(s):

N. RHIND ◽

B.A. BABER-FURNARI ◽

A. LOPEZ-GIRONA ◽

M.N. BODDY ◽

J.-M. BRONDELLO ◽

...

Keyword(s):

Dna Damage ◽

Fission Yeast ◽

Dna Damage Checkpoint ◽

Checkpoint Control

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Recruitment of DNA Damage Checkpoint Proteins to Damage in Transcribed and Nontranscribed Sequences

Molecular and Cellular Biology ◽

10.1128/mcb.26.1.39-49.2006 ◽

2006 ◽

Vol 26 (1) ◽

pp. 39-49 ◽

Cited By ~ 48

Author(s):

Guochun Jiang ◽

Aziz Sancar

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Excision Repair ◽

Dna Lesions ◽

Human Cells ◽

Dna Damage Checkpoint ◽

Damage Site ◽

Checkpoint Response ◽

Checkpoint Proteins ◽

Transcription Complexes

ABSTRACT We developed a chromatin immunoprecipitation method for analyzing the binding of repair and checkpoint proteins to DNA base lesions in any region of the human genome. Using this method, we investigated the recruitment of DNA damage checkpoint proteins RPA, Rad9, and ATR to base damage induced by UV and acetoxyacetylaminofluorene in transcribed and nontranscribed regions in wild-type and excision repair-deficient human cells in G1 and S phases of the cell cycle. We find that all 3 damage sensors tested assemble at the site or in the vicinity of damage in the absence of DNA replication or repair and that transcription enhances recruitment of checkpoint proteins to the damage site. Furthermore, we find that UV irradiation of human cells defective in excision repair leads to phosphorylation of Chk1 kinase in both G1 and S phase of the cell cycle, suggesting that primary DNA lesions as well as stalled transcription complexes may act as signals to initiate the DNA damage checkpoint response.

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