scholarly journals Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling

2006 ◽  
Vol 173 (2) ◽  
pp. 181-186 ◽  
Author(s):  
Shan Yan ◽  
Howard D. Lindsay ◽  
W. Matthew Michael
Keyword(s):  
Sperm Chromatin ◽  
Checkpoint Control ◽  
Direct Role ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Egg Extracts ◽  
Oligonucleotide Duplex ◽  
Stalled Replication Forks

TopBP1-like proteins, which include Xenopus laevis Xmus101, are required for DNA replication and have been linked to replication checkpoint control. A direct role for TopBP1/Mus101 in checkpoint control has been difficult to prove, however, because of the requirement for replication in generating the DNA structures that activate the checkpoint. Checkpoint activation occurs in X. laevis egg extracts upon addition of an oligonucleotide duplex (AT70). We show that AT70 bypasses the requirement for replication in checkpoint activation. We take advantage of this replication-independent checkpoint system to determine the role of Xmus101 in the checkpoint. We find that Xmus101 is essential for AT70-mediated checkpoint signaling and that it functions to promote phosphorylation of Claspin bound Chk1 by the ataxia-telangiectasia and Rad-3–related (ATR) protein kinase. We also identify a separation-of-function mutant of Xmus101. In extracts expressing this mutant, replication of sperm chromatin occurs normally; however, the checkpoint response to stalled replication forks fails. These data demonstrate that Xmus101 functions directly during signal relay from ATR to Chk1.

scholarly journals DDK has a primary role in processing stalled replication forks to initiate downstream checkpoint signaling

2017 ◽  
Author(s):  
Nanda Kumar Sasi ◽  
Flavie Coquel ◽  
Yea-Lih Lin ◽  
Jeffrey P MacKeigan ◽  
Philippe Pasero ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Primary Role ◽  
Replication Forks ◽  
M Cell ◽  
Replication Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Cycle Arrest ◽  
Stalled Replication Forks

AbstractCDC7-DBF4 kinase (DDK) is required to initiate DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has also been reported to have diverse and sometimes conflicting roles in the replication checkpoint response in various organisms but the underlying mechanisms are far from settled. Here we show that human DDK promotes limited resection of newly synthesized DNA at stalled replication forks or sites of DNA damage to initiate replication checkpoint signaling. DDK is also required for efficient fork restart and G2/M cell cycle arrest. DDK exhibits genetic interactions with the ssDNA exonuclease EXO1, and we show that EXO1 is also required for nascent strand degradation following exposure to HU, raising the possibility that DDK regulates EXO1 directly. Thus, DDK has a primary and previously undescribed role in the replication checkpoint to promote ssDNA accumulation at stalled forks, which is required to initiate a robust checkpoint response and cell cycle arrest to maintain genome integrity.

scholarly journals Undamaged DNA Transmits and Enhances DNA Damage Checkpoint Signals in Early Embryos

2007 ◽  
Vol 27 (19) ◽  
pp. 6852-6862 ◽  
Author(s):  
Aimin Peng ◽  
Andrea L. Lewellyn ◽  
James L. Maller
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Nuclear Dna ◽  
Cell Cycle Checkpoint ◽  
Dna Damage Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Egg Extracts ◽  
Cycle Checkpoint ◽  
Damaged Dna

ABSTRACT In Xenopus laevis embryos, the midblastula transition (MBT) at the 12th cell division marks initiation of critical developmental events, including zygotic transcription and the abrupt inclusion of gap phases into the cell cycle. Interestingly, although an ionizing radiation-induced checkpoint response is absent in pre-MBT embryos, introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. We show here that undamaged threshold DNA directly participates in checkpoint signaling, as judged by several dynamic changes, including H2AX phosphorylation, ATM phosphorylation and loading onto chromatin, and Chk1/Chk2 phosphorylation and release from nuclear DNA. These responses on physically separate threshold DNA require γ-H2AX and are triggered by an ATM-dependent soluble signal initiated by damaged DNA. The signal persists in egg extracts even after damaged DNA is removed from the system, indicating that the absence of damaged DNA is not sufficient to end the checkpoint response. The results identify a novel mechanism by which undamaged DNA enhances checkpoint signaling and provide an example of how the transition to cell cycle checkpoint activation during development is accomplished by maternally programmed increases in the DNA-to-cytoplasm ratio.

scholarly journals TopBP1 and DNA polymerase-α directly recruit the 9-1-1 complex to stalled DNA replication forks

2009 ◽  
Vol 184 (6) ◽  
pp. 793-804 ◽  
Author(s):  
Shan Yan ◽  
W. Matthew Michael
Keyword(s):  
Dna Polymerase ◽  
Replication Stress ◽  
Replication Forks ◽  
Ataxia Telangiectasia Mutated ◽  
Interacting Protein ◽  
Checkpoint Signaling ◽  
Binding Partner ◽  
Assembly Pathway ◽  
Egg Extracts ◽  
Stalled Replication Forks

TopBP1 and the Rad9–Rad1–Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR–ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-α to sites of replication stress. Furthermore, we show that pol-α is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-α, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.

scholarly journals DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7

2004 ◽  
Vol 167 (5) ◽  
pp. 841-849 ◽  
Author(s):  
Ayumi Yamada ◽  
Brad Duffy ◽  
Jennifer A. Perry ◽  
Sally Kornbluth
Keyword(s):  
Dna Replication ◽  
Cell Cycle Control ◽  
Checkpoint Control ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Synthetic Lethal ◽  
Mitotic Entry ◽  
Regulatory Module ◽  
Dna Replication Checkpoint ◽  
Control Modules

G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7–Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organsims.

scholarly journals The Xenopus Chk1 Protein Kinase Mediates a Caffeine-sensitive Pathway of Checkpoint Control in Cell-free Extracts

1998 ◽  
Vol 142 (6) ◽  
pp. 1559-1569 ◽  
Author(s):  
Akiko Kumagai ◽  
Zijian Guo ◽  
Katayoon H. Emami ◽  
Sophie X. Wang ◽  
William G. Dunphy
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Checkpoint Control ◽  
Checkpoint Response ◽  
Cell Cycle Delay ◽  
Checkpoint Pathway ◽  
Egg Extracts ◽  
A Cell

We have analyzed the role of the protein kinase Chk1 in checkpoint control by using cell-free extracts from Xenopus eggs. Recombinant Xenopus Chk1 (Xchk1) phosphorylates the mitotic inducer Cdc25 in vitro on multiple sites including Ser-287. The Xchk1-catalyzed phosphorylation of Cdc25 on Ser-287 is sufficient to confer the binding of 14-3-3 proteins. Egg extracts from which Xchk1 has been removed by immunodepletion are strongly but not totally compromised in their ability to undergo a cell cycle delay in response to the presence of unreplicated DNA. Cdc25 in Xchk1-depleted extracts remains bound to 14-3-3 due to the action of a distinct Ser-287-specific kinase in addition to Xchk1. Xchk1 is highly phosphorylated in the presence of unreplicated or damaged DNA, and this phosphorylation is abolished by caffeine, an agent which attenuates checkpoint control. The checkpoint response to unreplicated DNA in this system involves both caffeine-sensitive and caffeine-insensitive steps. Our results indicate that caffeine disrupts the checkpoint pathway containing Xchk1.

scholarly journals Colocalization of Mec1 and Mrc1 is sufficient for Rad53 phosphorylation in vivo

2012 ◽  
Vol 23 (6) ◽  
pp. 1058-1067 ◽  
Author(s):  
Theresa J. Berens ◽  
David P. Toczyski
Keyword(s):  
Dna Replication ◽  
Double Strand Breaks ◽  
Sensor Kinase ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Strand Breaks ◽  
Crucial Step ◽  
Stalled Replication Forks

When DNA is damaged or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore we constructed an in vivo replication-checkpoint mimic that recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the endogenous replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires colocalization of Mrc1-LacI and Ddc2-LacI and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the endogenous replication checkpoint but that Ddc1 is absolutely required in the absence of Mrc1. We propose that colocalization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.

The DNA replication checkpoint response stabilizes stalled replication forks

Nature ◽  
2001 ◽  
Vol 412 (6846) ◽  
pp. 557-561 ◽  
Author(s):  
Massimo Lopes ◽  
Cecilia Cotta-Ramusino ◽  
Achille Pellicioli ◽  
Giordano Liberi ◽  
Paolo Plevani ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Forks ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
Dna Replication Checkpoint ◽  
Stalled Replication Forks

scholarly journals MDC1 collaborates with TopBP1 in DNA replication checkpoint control

2011 ◽  
Vol 193 (2) ◽  
pp. 267-273 ◽  
Author(s):  
Jiadong Wang ◽  
Zihua Gong ◽  
Junjie Chen
Keyword(s):  
Dna Replication ◽  
Cellular Response ◽  
Checkpoint Control ◽  
Dna Double Strand Breaks ◽  
Replication Checkpoint ◽  
Strand Breaks ◽  
Major Player ◽  
Dna Replication Checkpoint ◽  
Cellular Dna ◽  
Stalled Replication Forks

Human TopBP1 is a major player in the control of the DNA replication checkpoint. In this study, we identified MDC1, a key checkpoint protein involved in the cellular response to DNA double-strand breaks, as a TopBP1-associated protein. The specific TopBP1–MDC1 interaction is mediated by the fifth BRCT domain of TopBP1 and the Ser-Asp-Thr (SDT) repeats of MDC1. In addition, we demonstrated that TopBP1 accumulation at stalled replication forks is promoted by the H2AX/MDC1 signaling cascade. Moreover, MDC1 is important for ATR-dependent Chk1 activation in response to replication stress. Collectively, our data suggest that MDC1 facilitates several important steps in both cellular DNA damage response and the DNA replication checkpoint.

scholarly journals The CDK-PLK1 axis targets the DNA damage checkpoint sensor protein RAD9 to promote cell proliferation and tolerance to genotoxic stress

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Takeshi Wakida ◽  
Masae Ikura ◽  
Kenji Kuriya ◽  
Shinji Ito ◽  
Yoshiharu Shiroiwa ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Cell Cycle Progression ◽  
Genotoxic Stress ◽  
Dna Damage Checkpoint ◽  
Proliferating Cells ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Promote Cell Proliferation

Genotoxic stress causes proliferating cells to activate the DNA damage checkpoint, to assist DNA damage recovery by slowing cell cycle progression. Thus, to drive proliferation, cells must tolerate DNA damage and suppress the checkpoint response. However, the mechanism underlying this negative regulation of checkpoint activation is still elusive. We show that human Cyclin-Dependent-Kinases (CDKs) target the RAD9 subunit of the 9-1-1 checkpoint clamp on Thr292, to modulate DNA damage checkpoint activation. Thr292 phosphorylation on RAD9 creates a binding site for Polo-Like-Kinase1 (PLK1), which phosphorylates RAD9 on Thr313. These CDK-PLK1-dependent phosphorylations of RAD9 suppress checkpoint activation, therefore maintaining high DNA synthesis rates during DNA replication stress. Our results suggest that CDK locally initiates a PLK1-dependent signaling response that antagonizes the ability of the DNA damage checkpoint to detect DNA damage. These findings provide a mechanism for the suppression of DNA damage checkpoint signaling, to promote cell proliferation under genotoxic stress conditions.

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